The induction of DNA synthesis in the chick red cell nucleus in heterokaryons during the first cell cycle after fusion with HeLa cells.

Induction of DNA synthesis in embryonic chick red cells has been examined during the first and second cell cycles after fusion with HeLa cells synchronized in different parts of G1 and S-phase. The data indicate that: (i) the younger the embryonic blood the more rapidly the red cells are induced into DNA synthesis; (ii) the greater the ratio of HeLa to chick nuclei in the heterokaryon, the more rapidly the induction occurs; (iii) DNA synthesis in the chick nucleus can continue after the HeLa nucleus has left S-phase and entered either G2 or mitosis; (iv) the induction potential of late S-phase HeLa is somewhat lower than that of early or mid S-phase cells; (v) less than 10% of the chick DNA is replicated during the first cycle after fusion and only a small proportion (15%) of the chick nuclei approach the 4C value of DNA during the second cycle after fusion; (vi) the newly synthesized DNA is associated either with the condensed regions of the nucleus or with the boundaries between condensed and non-condensed regions; (vii) the chick chromosomes at the first and second mitosis after fusion are in the form of PCC prematurely condensed chromosomes); they are never fully replicated and are often fragmentary; (viii) DNA synthesis in the chick nuclei is accompanied by an influx of protein (both G1 and S-phase protein) from the HeLa component of the heterokaryon.     

Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha.     

Haploid genome reactivation and recovery by cell hybridization

DNA replication in haploid spermatid nuclei has been induced by hybridization of mouse early spermatids to proliferating HeLa cells. Use of polyethylene glycol rather than inactivated Sendai virus as the cell fusion agent was found to be essential to the production of large numbers of heterokaryons containing spermatid nuclei. DNA replication was detected in the heterokaryons by autoradiography. Density of silver grains over spermatid nuclei closely approximated the grain density over labelled HeLa nuclei in the same heterokaryons. Mouse centromeric heterochromatin appeared to be labelled last during the spermatid DNA synthetic period. On the average, HeLa nuclei in heterokaryons began DNA synthesis before spermatid nuclei. Results indicated, however, that DNA synthesis by HeLa nuclei might not be a prerequisite for spermatid DNA synthesis. These experiments demonstrate induction of DNA synthesis in spermatid nuclei, the first major step toward reactivation and recovery of their haploid genome by cell hybridization.     

Factors influencing ADP-ribosylation differences between chromosomal proteins of interphase and metaphase HeLa cells

Fundamental differences were previously discovered in the ADP-ribosylation of proteins from metaphase chromosomes and interphase nuclei of HeLa cells. The number of modified nonhistone species was found to be dramatically reduced for metaphase chromosomes. An investigation has therefore been made of factors which could influence, and therefore be responsible for, this change in ADP-ribosylation during the cell cycle. Modified proteins were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing mitotic and interphase samples from permeabilized cells that had been incubated with [32P]NAD. Whole cells showed a difference between interphase and metaphase similar to that for isolated nuclei and chromosomes. Chromosome expansion, disruption of chromosomes or nuclei, DNA nicking, and cellular growth activity significantly changed the incorporation of 32P label. Inhibitors of protein, RNA, and DNA synthesis did not, however, greatly affect ADP-ribosylation. The pattern of labeled species was not altered by the presence of nonradioactive NAD, though the extent of labeling declined. The results were not artifactually due to the procedure used to arrest cells in mitosis. Similar results were found with Novikoff rat hepatoma cells, demonstrating that the difference between metaphase and interphase is not confined to HeLa cells.     

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA.     

Initiation of DNA Synthesis in HeLa Cell-free System

THE molecular mechanism for initiating DNA replication can be studied using a subcellular system. Rao and Johnson¹ found that HeLa cells in the pre-DNA-synthetic (G-1) period of the cell cycle initiate DNA synthesis after fusion with cells that are in the DNA synthetic (S) period. A previous subcellular system of DNA replication from HeLa cells^2–4 consisted of intact nuclei, supplemented with the four deoxy-nucleoside triphosphates, salt, ATP and a cytosol factor. The nuclei in this system appeared to be permeable to proteins and DNA synthesis was very similar to that within intact cells. We report here the initiation of DNA synthesis in nuclei isolated from HeLa cells. Our results suggest that, with the synchronization method used, a small percentage of dormant G-1 nuclei can be stimulated by S-phase cytoplasm; this would be the case if the cells were receptive to stimulation for only 30–60 min during the cell cycle. illustration

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Nanomolar concentrations of untransformed glucocorticoid receptor in nuclei of intact cells

The subcellular distribution of untransformed glucocorticoid-receptor complex in vivo has been studied by chemical crosslinking of intact cells, and using a procedure adequate for correction of experimental errors due to redistribution of components between cytosolic and nuclear fractions. We found that in HeLa S₃ cells 85.4% of total glucocorticoid-receptor complexes are located in nuclei, and 14.6% are cytosolic. If measurements were performed with MCF-7 cells, we determined that the nuclear pool of glucocorticoid-receptor complexes accounts for 75.2% of the total cellular content, whereas the remaining 24.8% are cytosolic. When the subcellular distribution of estrogen-receptor complexes was determined, instead, we found that they are almost exclusively located in nuclei of MCF-7 cells, which contain 88.9% of the total. In order to estimate the molar concentration of receptors in cytosol and nuclei of intact cells, we determined the free water content of the two compartments. The volume of solvent was found to vary in the three cell lines we have studied, and our data showed that these variations are due to the cytosolic fractions, as the free water content of nuclei is essentially the same in those cells. When the free water content and the levels of glucocorticoid-receptor complexes we have measured were used to estimate the molar ncentrations of receptors, we found that these range between 0.4 and 18.9 nM in cytosols, and between 3.9 and 6.3 nM in nuclei of the three cell lines we have studied. We then concluded that the relative distribution of untransformed glucocorticoid-receptor complexes between cytosol and nuclei is cell-specific but their molar concentration in the nuclear compartment does not greatly vary among different cells.     

A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.     

Poly adenylic acid synthesis in vitro in isolated HeLa cell nuclei and whole cell homogenates,

Homogenates of HeLa cells and purified HeLa cell nuclei were found to synthesize poly(A) in vitro. The poly(A) is the normal adenylate-rich species seen in growing cells and is contained in large RNA molecules, which themselves have been synthesized at least in part in vitro. Poly(A) synthesis in purified nuclei shows a dependence on ATP concentration that is about 75–200 times higher than the concentration for total RNA synthesis.     

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.     

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin.

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drug's ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.     

Poly(ADP-ribose) synthesis and cell division

A clone of HeLa cells has been selected in the presence of 5-methylnicotinamide. In the presence of 10 mM 5-methylnicotinamide the resistant cells grow at 70% of the rate of the same cell culture without 5-methylnicotinamide. 10 mM 5-methylnicotinamide completely inhibits the growth of normal HeLa cells. Both cell types in the absence of 5-methylnicotinamide have the same generation time. Poly (adenosine diphosphate-ribose) synthesis is less sensitive to 5-methylnicotinamide in nuclei isolated from the resistant cells than in nuclei from sensitive cells.     

Localization of non-native D-glyceraldehyde-3-phosphate dehydrogenase in growing and apoptotic HeLa cells

Monoclonal antibodies that could not bind native tetramers of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) but could bind to dimeric, monomeric, or denatured forms of GAPDH were used to investigate its intracellular localization. These antibodies distinctly stained the nucleus in growing HeLa cells. In the cytoplasm, non-native GAPDH was colocalized with actin filaments. Incubation of HeLa cells with tumor necrosis factor α (TNF-α) and the protein synthesis inhibitor emetine led to a drastic increase in the amount of the non-native GAPDH in the nuclei. Overproduction of Bcl-2 protein did not change the non-native GAPDH localization in the growing HeLa cells but prevented the development of apoptosis and the increase in the amount of non-native GAPDH in the nuclei upon incubation with TNF-α.     

Changes in eIF-4D hypusine modification or abundance are not correlated with translational repression in HeLa cells

Initiation factor eIF-4D is represented by about 11 X 10(6) molecules/HeLa cell (0.45% of the cytoplasmic protein molecules). The fraction of eIF-4D that contains the post-translational modification of lysine converted to hypusine is not regulated with respect to translation rate in HeLa cells. It is proportional to the rate of eIF-4D synthesis in exponentially growing cells (maximal protein synthesis rates) as well as in serum-depleted cells (protein synthesis rates depressed about 6-8-fold). In cells in which protein synthesis is arrested by cycloheximide, no hypusine addition or exchange is detected. During rapid repressions of protein synthesis due to either heat shock or hypertonic shock there is no change in the extent of eIF-4D containing hypusine. These results are most consistent with an eIF-4D biogenesis in which all molecules are modified to contain hypusine during or shortly after the translation process itself, and the modification state is not regulated thereafter.     

Inhibition of protein synthesis in reovirus-infected HeLa cells with elevated levels of interferon-induced protein kinase activity

Protein synthesis was inhibited in one line of interferon-treated HeLa cells (line 2) upon infection with reovirus, but not in different HeLa cells (line 1) treated in the same way. The inhibition resulted in polysome runoff, suggesting that it was due to an impairment of peptide chain initiation. Interferon induces the synthesis of a protein kinase, which is activated in cell-free systems by double-stranded RNA and phosphorylates the alpha subunit of eukaryotic initiation factor 2, thus inhibiting the initiation of protein synthesis. Therefore, we measured the level of this protein kinase in extracts prepared from the two HeLa cell lines. Cells of line 2 showed about 3-4 times more protein kinase activity than cells of line 1. The inhibition of protein synthesis upon infection with reovirus was correlated with an increased phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in interferon-treated cells labeled with 32P. The kinase was presumably activated in intact cells by viral double-stranded RNA, but this activation resulted in inhibition of protein synthesis only in cells with elevated levels of the kinase.     

HeLa cell nucleus, a source of thymidine for Trichomonas vaginalis growing in vitro

Trichomonas vaginalis is a parasitic protist incapable of de novo purine and pyrimidine biosynthesis. The lack of these de novo syntheses of nucleotides is supplemented with purine and pyrimidine salvage pathways. Likewise, T. vaginalis is incapable of converting its ribonucleotides to deoxyribonucleotides. Therefore, the parasite must rely on the salvage of exogenous deoxyribonucleosides for DNA synthesis. It has been demonstrated that the parasite can incorporate external adenine and guanine in vitro, but no in vivo nucleotide source has been identified so far. Accordingly, we set out to determine if the parasite could incorporate 3H-thymidine from the nuclei of a cervical-derived cell line into its own DNA. By light and electron microscopy we found that the parasite was able to interact directly, both with mechanically isolated HeLa cell nuclei and with the nuclei released after the disruption of HeLa cell monolayers by the parasite. This study shows that T. vaginalis was capable of incorporating 3H-thymidine from labeled HeLa cells into its own DNA suggesting that the nuclei of this cervical cell line could be an in vivo source of nucleotides for T. vaginalis.     

Protein synthesis is not required for the inhibitory effect of selenite on cell colony formation and RNA synthesis

Selenite has been shown to undergo intracellular metabolism that results in its conversion to other low molecular weight Secontaining species and also to its incorporation into a selenocysteine residue in selenoprotein. In order to investigate whether the incorporation into protein is required for the cytotoxic effects of selenite, we have examined whether inhibition of protein synthesis prevents the inhibitory effect of selenite on the ability of cells to form colonies or to synthesize RNA. We have found that treatment of HeLa cells with cycloheximide inhibited protein synthesis by >90% but had no effect on the inhibitory effect of selenite on cell colony formation or RNA synthesis. Since protein synthesis is not necessary for these cytotoxic effects of selenite they are unlikely to result from an increase in the synthesis of selenoproteins.     

Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells.

The nonhistone chromosomal protein HMG1 associated rapidly with the nuclei of HeLa cells and bovine fibroblasts following its introduction into the cytoplasm by red cell-mediated microinjection. A number of non-nuclear proteins, on the other hand, failed to concentrate in HeLa or bovine fibroblast nuclei. Autoradiography of thin sections showed that 125I-labeled HMG1 localized within nuclei, and further established that it remained associated with metaphase chromosomes at mitosis. When uninjected HeLa cells were fused with 125I-HMG1-injected HeLa cells, the labeled molecules equilibrated between nuclei within 12 hr. Similar results were obtained with bovine fibroblasts, indicating that a dynamic equilibrium exists between HMG1 and chromatin within living cells. Electrophoresis of 125I-HMG1 retrieved from HeLa cells or bovine fibroblasts up to 48 hr after injection showed that more than 80% of the molecules were intact. Autoradiographic analysis of cells fixed over a period of several days after injection produced apparent half-lives for 125I-HMG1 of 80 hr in HeLa cells and 100 hr in bovine fibroblasts.