Regulation of DNA synthesis in cytochalasin B (CB)-induced binucleate HeLa cells

The effects of timing and duration of cytochalasin B (CB) treatment on the kinetics of the initiation of DNA synthesis in mono- and binucleate HeLa cells, synchronized in the G1 phase of the cell cycle by the reversal of a mitotic block (N₂O at 80 PSI), were studied. In the control, bi-, tri- and tetranucleate cells entered S phase slightly earlier than the mononucleate cells at a rate proportional to the number of their nuclei. The difference between any two adjacent sub-populations was less than 0.5 h. However, the binucleate cells produced by a 90 min CB treatment immediately after the reversal of the mitotic block exhibited a considerably shorter G1 period as compared to mononucleate cells (a difference of 1.5 h). This exaggerated difference in the duration of G1 period between mono- and binucleate cells disappeared when the CB treatment was delayed by 75 or 90 min indicating that it was an experimental artifact. From this study, we conclude that there is naturally some degree of nuclear cooperation in the multinucleate systems, particularly with regard to the initiation of DNA synthesis, which is not influenced by CB treatment.     

Normal level of unscheduled DNA synthesis in Werner''s syndrome fibroblasts in culture

We investigated UV-induced unscheduled DNA synthesis (UDS) in skin fibroblasts from seven unrelated patients with clinically apparent Werner's syndrome (WS). WS cells exhibited greatly abbreviated in vitro lifespans, the extents of which ranged from about 20 to 50% of the normal. However, WS cells in early and senescent phases of growth showed the same quantity of DNA repair following UV exposure as did normal fibroblasts.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. I. Composition and metabolism of nonhistone chromosomal proteins.

The composition and metabolism of nonhistone chromosomal proteins associated with the genome of normal and SV40 transformed WI38 human diploid fibroblasts were examined. Evidence is presented which indicates variations in the relative protein content, specific activity and phosphorylation of several defined molecular weight classes of these chromosomal polypeptides. In addition, it is shown that blocking DNA replication with cytosine arabinoside does not inhibit the incorporation of ³H-leucine into nonhistone chromosomal proteins of SV40 transformed cells. The possibility that the nonhistone chromosomal proteins of human diploid fibroblasts transformed by SV40 virus may be involved in the regulation of viral induced alterations in gene expression is discussed.     

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.     

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.     

Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis.

ts Cl mouse L cells are temperature-sensitive (ts) in DNA synthesis. The protein involved undergoes inactivation at 38.5 °C, with an apparent half-life of 3–4 h. A variety of experimental approaches yield data indicating that the ts Cl gene product acts directly during the DNA-synthesis period, probably late during the duplication of chromosomal DNA. The specificity of the ts lesion is reflected in the fact that replication of mitochondrial DNA is unaffected for many hours after nuclear DNA synthesis is almost totally inhibited. Temperature inactivation is not due to degradation or to loss of template capacity of preformed DNA. ts Cl cells are able to enter a DNA-synthesis phase at the higher temperature, as indicated by radioautographic experiments and by studies in which cells, blocked at the permissive temperature (34 °C) in a pre-DNA synthesis phase by isoleucine deprivation, are subsequently incubated at 38.5 °C. Cells arrested early in DNA synthesis by hydroxyurea treatment at 34 °C continue such synthesis for a short interval after up-shift to 38.5 °C. However, they are then unable to complete the S phase in progress nor can they proceed into cell division. The kinetics of DNA synthesis in cells incubated at 38.5 °C and back-shifted to 34 °C are compatible with the model that the ts Cl locus encodes an S phase function.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Comparison of pyridoxal phosphate and 0.4 M KCl-extracted nuclear glucocorticoid receptors in HeLa S3 cells

A comparison of the physicochemical properties between pyridoxal 5'-phosphate- and 0.4 M KCl-extracted nuclear glucocorticoid receptors has been made utilizing HeLa S3 cells as a source of receptor. Both pyridoxal 5'-phosphate/NaBH4-reduced and 0.4 M KCl-extracted receptors sedimented as approximately 3.5-4.5 S species in 5-20% sucrose gradients containing 0, 0.15, and 0.4 M KCl. Under low-ionic-strength buffer conditions, pyridoxal 5'-phosphate-extracted receptor elutes close to the void volume of a Sephacryl S-300 gel-exclusion column. Increasing the [KCl] of the column to 0.4 M resulted in the elution of receptor with a Stokes radius of 58 A and calculated Mr = 96,000. Nuclear receptors extracted with 0.4 M KCl also formed a large-molecular-weight complex which eluted close to the void volume of the gel-exclusion column. Increasing the [KCl] to 0.4 M had the effect of shifting this receptor form to a species which had a Stokes radius of 62 A and calculated Mr = 89,700. Ion-exchange analysis of nuclear-extracted receptors revealed that 0.4 M KCl-extracted receptors exhibited considerable charge heterogeneity, whereas pyridoxal 5'-phosphate-extracted receptors did not. Pyridoxal 5'-phosphate-extracted receptors (approximately 86%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M; approximately 14% of the receptors had little affinity for DEAE-cellulose. Pyridoxal phosphate-treated receptors had little affinity for hydroxylapatite, phosphocellulose, and DNA-cellulose. The predominant form of 0.4 M KCl-extracted nuclear receptors (approximately 78%) eluted from DEAE-cellulose between 0.05 and 0.15 M KCl, a position coincident with "activated" glucocorticoid receptors. The remaining receptor fraction (approximately 22%) eluted from DEAE-cellulose at a [KCl] greater than 0.15 M, a position coincident with "unactivated" glucocorticoid receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of silicon in diatom metabolism. VII. Silicic acid-stimulated DNA synthesis in toluene-permeabilized cells of Cylindrotheca fusiformis.

Human diploid cell populations were fractionated on the basis of cell size by gravity sedimentation. This cell separation procedure yielded fractionated cell populations that were enriched for both large cell volumes and for slow and/or non-replicating cells (cells which did not have labeled nuclei after a 48 h incubation with [³H]TdR). It also yielded fractionated cell populations that were enriched for small cell volumes and for rapidly replicating cells (cells with labeled nuclei). However, upon reintroduction to tissue culture conditions, cell populations lost their fractionated properties and soon resembled unfractionated cell cultures at similar levels of in vitro passage.     

Immunofluorescent and autoradiographic analysis of the relationship between the presence of histone H5 and DNA synthesis in developing chick erythroid cells

Simultaneous detection of histone H5 by indirect immunofluorescence and of [³H]thymidine incorporation by autoradiography on the same preparations of developing erythroid cells have been used to precisely define the extent of correlation between the loss of nuclear activity and the presence of histone H5. It was found that from day 3–12 of embryonic life there are two successive waves of double-labelled cells. At some stages, as many as 30% of the cells which incorporate [³H]thymidine also contain histone H5. Thus, the simple presence of H5 cannot be sufficient to cause nuclear inactivation. A kinetic analysis of the appearance and disappearance of [³H]thymidine-labelled cells, containing histone H5, and cells which are positive for both markers is presented. The result is consistent with the interpretation that the appearance of H5 in the first wave of double labelled cells occurs just before the erythroid cells become metabolically inactive. These observations modify the concept that histone H5 functions uniquely or solely as a template repressor.     

Activity of lysosomal enzymes in the various cell cycle phases of synchronized HeLa cells.

The activities of 5 lysosomal enzymes (acid DNase, β-glucuronidase, β-N-acetylglucosaminidase, β-galactosidase and cathepsin D) were measured in HeLa cells in various cell cycle phases. The cells were synchronized either by shake-off of mitotic cells followed by resuspension in fresh medium, or by addition of amethopterin and adenosine for 16 h and reversal with thymidine. Metaphase arrest was obtained with colcemid in cells previously synchronized by means of amethopterin/thymidine. The specific activities (activity/mg protein) of the different enzymes were found to be constant following synchronization both with the shake-off technique and with the amethopterin/thymidine treatment. Furthermore, the specific enzyme activities were unaltered by metaphase arrest by colcemid. Our data indicate that lysosomal enzyme synthesis is continuous during the cell cycle of HeLa cells. The specific activity of β-glucuronidase was found to be about 3 times higher in HeLa cells grown in suspension cultures than in cells grown on solid surface. The activities of the other enzymes measured were approximately equal in suspension cells and surface cells.     

Correlation of (Na---K)-ATPase activity with growth of normal and transformed cells

The (Na⁺---K⁺)-stimulated Mg²⁺-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na⁺---K⁺)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na⁺---K⁺)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.     

Production of gamma interferon by human T and null cells and its regulation by macrophages

Human mononuclear subpopulations were tested for the capacity to produce interferon after mitogenic stimulation with protein A from Staphylococcus aureus. Mononuclear cells were separated into highly enriched macrophage, T-, B-, and null-cell subpopulations by Sephadex G-10 adherence, anti-human IgG F(ab′) two-column chromatography, and rosetting with sheep erythrocytes. Interferon (IFN) production was observed in both T- and null-cell preparations, but not in macrophage or B-cell preparations. Physicochemical and antigenic characterization of IFN from T- and null-cell preparations showed that both mononuclear subpopulations produced gamma IFN (IFNγ). Regulatory studies showed that IFNγ production was differentially regulated by macrophages. Macrophage addition to T lymphocytes augmented both cellular proliferation and IFNγ production, whereas macrophage addition to null cells suppressed IFNγ production and had no effect on the minimal proliferative response observed for these cells.     

δ-aminolevulinic acid transport and synthesis,porphyrin synthesis and heme catabolism in chick embryo liver and heart cells

Liver and heart represent two organs with markedly different needs for heme as related to their metabolic roles. To examine these diferences chick embryo heart and liver cells were compared with respect to transport of δ-aminolevulinic acid and activity of δ-aminolevulinic acid synthetase, porphyrin synthesis and heme oxygenase. Heart cells were found to have a low rate of δ-aminolevulinic acid uptake, a high resting level of δ-aminolevulinic acid synthetase activity and a lower level of heme oxygenase activity as compared with liver cells. The hepatic cell uptake of δ-aminolevulinic acid was 6–25-times that of heart cells. The embryonal heart cell appears to be a balanced autonomous system for the synthesis and degradation of heme. The embryonal liver cell represents a cell system permeable to exogenous δ-aminolevulinic acid, which is also responsive to and inducible by external stimuli.     

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.     

Evidence for selection in cultured diploid fibroblast strains

Single skin fibroblast cultures were established from 29 females heterozygous at the X-linked G-6-PD locus for Gd^B and an electrophoretic variant, Gd^A or Gd^A?. The cultures were transferred serially until they displayed single-enzyme phenotypes (only type A or type B enzyme) or until growth ceased. Single-enzyme phenotypes were observed in 19 instances. This occurred as early as the 4th, and as late as the 38th transfer. Ten cultures evolved A phenotypes and 9 developed B phenotypes. Possible explanations for the development of single-enzyme phenotypes include random cell-sampling during sub-culturing, selection or both. Selection occurring at least in part on the basis of X-linked loci other than G-6-PD seems most likely for those cultures which evolved rapidly to single-enzyme phenotypes, but sampling or selection or both could have played important roles in those cultures which maintained double-enzyme phenotypes for several passages and then rapidly developed single-enzyme phenotypes. In any event, the results indicate that it should not be assumed in investigations using cultivated diploid fibroblasts that the latter are generally representative of the tissue-as-a-whole from which the cultures originate.