Fast repair of anoxic radiation damage in HeLa cells detected by antinucleoside antibodies

The influence of anoxia on X-ray damage in HeLa cells was studied by observing effects on nuclear immunoreactivity to antinucleoside antibodies and on the sedimentation in alkaline sucrose gradients of their DNA “complexes”. The fraction of G1 HeLa cells which was immunoreactive to fluorescein labeled antinucleoside antibodies increased from control levels of 11% ± 3.5 S.E. to 71% ± 5.7 S.E. after 1 000 rads in air. In anoxia 1 000 rads increased this fraction to only 42% ± 3.1 S.E. After 1 000 rads in air the return to normal G1 levels of immunoreactivity required 90 min, but it required only 30 min after radiation in anoxia. If cells were held at 0 °C for 35 min before anoxic irradiation the rapid return to control levels of immunoreactivity during postradiation incubation at 37 °C was not observed. Cold shock did not increase the proportion of cells initially made immunoreactive by 1 000 rads in anoxia. Anoxia reduced the effect of 1 000 rads on the sedimentation properties of the DNA complex. Cold shock prior to anoxic radiation retarded the faster reconstitution of the DNA complex otherwise observed after anoxic radiation.     

Immunoreactivity to antinucleoside antibodies in camptothecin treated HeLa cells

HeLa cells, incubated with camptothecin during the G 1 phase of the cell cycle, show nuclear fluorescence with fluorescein-labeled antinucleoside antibodies. If the G 1 cells are washed free of the drug, the cells no longer demonstrate nuclear fluorescence. Since these antibodies react only with single-stranded DNA, the positive staining in camptothecin-treated G 1 cells suggests that the drug induces denatured regions in DNA. Fluorescent antinucleoside antibodies may be a useful technique for the observation of drug-induced changes in DNA during the G1 phase of the cell cycle.     

A STUDY OF THE PENETRATION OF MAMMALIAN CELLS BY DEOXYRIBONUCLEIC ACIDS

Tritium-labeled deoxyribonucleic acid (DNA) from pneumococci and from human leukocytes was added to growing cultures of HeLa cells at 37°C. Autoradiography revealed an extensive localization of tritium in the nuclear regions. The label could not be removed by treatment with ribonuclease or dilute perchloric acid, but quantitative removal from the cells could be effected with deoxyribonuclease. Chemical and radioactivity determinations on nucleic acids isolated from the exposed HeLa cells revealed the presence of tritium in all 4 DNA bases. About 12 µg. of tritiated DNA was recovered from 6 x 10⁶ HeLa cells which had been exposed for 24 hours to 240 µg. of the human DNA. From this, it is concluded that the amount of DNA, or its degradation products, taken up by the cells was equivalent to at least 10 per cent of the normal HeLa cell complement.     

Immunoreactivity to antinucleoside antibodies persists during G2 arrest in X-irradiated HeLa cells

Arrest of HeLa cells in G2 after ionizing radiation is accompanied by persistent nuclear immunoreactivity to antinucleoside antibodies. The reactivity declined to the normal G2 level during escape from arrest and subsequent cell division.     

Lack of effect of butyrate on S phase DNA synthesis despite presence of histone hyperacetylation

The effects of sodium butyrate on [³H]thymidine incorporation and cell growth characteristics in randomly growing and synchronized HeLa S₃ cells have been examined in an attempt to determine what effects, if any, butyrate has on S phase cells. Whereas 5 mM sodium butyrate rapidly inhibits [⁵H]thymidine incorporation in a randomly growing cell populations, it has no effect on incorporation during the S phase in cells synchronized by double thymidine block techniques. This lack of effect does not result from an impaired ability of the S phase cells to take up butyrate, since butyrate administration during this period leads to histone hyperacetylation that is identical with that seen with butyrate treatment of randomly growing cells. Furthermore, the ability to induce such hyperacetylation with butyrate during an apparently normal progression through S phase indicates that histone hyperacetylation probably has no effect on the overall process of DNA replication. Temporal patterns of [³H]thymidine incorporation and cell growth following release from a 24-h exposure to butyrate confirm blockage of cell growth in the G1 phase of the cell cycle. Thus, the inhibition by butyrate of [³H]thymidine incorporation in randomly growing HeLa S₃ cell populations can be accounted for solely on the basis of a G1 phase block, with no inhibitory effects on cells already engaged in DNA synthesis or cells beyond the G1 phase block at the time of butyrate administration.     

Release of template restriction for DNA synthesis by poly ADP(ribose) polymerase during the HeLa cell cycle.

The degree of complexing between DNA and chromosomal proteins and the ability of poly adenosine diphosphate ribosylation (ADP-ribosylation) of nuclear proteins to release this template restriction and expose DNA primer site changes during the HeLa cell cycle. Primer site exposure by NAD and poly ADP(ribose) polymerase was assessed with intact nuclei by single deoxynucleotide incorporation into DNA in the presence of saturating bacterial DNA polymerase. The most marked in vitro enhancement of primer site exposure by ADP-ribosylation occurred in early G1 phase, where cellular template restriction was the greatest. Cytoplasmic DNA polymerase also had high activity in early G1 phase of the cell cycle. Streptozotocin reduces NAD pools in HeLa cells; a concomitant stimulation of nuclear poly ADP(ribose) polymerase activity is noted.     

In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences.

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, we isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G.C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G.C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.     

Characterization of large mammalian DNA species sedimented in a reorienting zonal rotor

The characteristics of a Beckman-designed slow acceleration unit for the reorientation of alkaline sucrose gradients in a Ti-15 zonal rotor are described. The large DNA species (> 250S) obtained from cultured rat brain tumor cells with this system sediment linearly with time, have virtually no [³H]leucinelabeled or covalently bonded [³H]choline-labeled material sedimenting with them, sediment independently of smaller single-stranded DNA molecules (? 165S) and are 60–80% degraded by the single-strand-specific S1 nuclease. Therefore, it is postulated that these species are collapsed, partially denatured DNA molecules or a collapsed form of single-stranded DNA. When cells were labeled with [¹⁴C]TdR, then frozen and stored at ? 79°C, this system could detect radiation-induced DNA damage from decay of the incorporated label at accumulated doses as small as 18–126 rads.     

Isolation and characterization of cancer stem cells from cervical cancer HeLa cells

Cervical cancer is one of the most common gynecologic malignancies and poses a serious health problem worldwide. Identification and characterization of cervical cancer stem cells may facilitate the development of novel strategies for the treatment of advanced and metastatic cervical cancer. Breast cancer-resistance protein (Bcrp1)-positive cells were selected from a population of parent HeLa cells using flow cytometry. The invasion capacity of Bcrp1-positive and -negative cells was analyzed with a Boyden chamber invasion test. The tumorigenicity of these cells was determined by in vivo transplantation in non-obesity diabetes/severe combined immunodeficiency (NOD/SCID) mice. The Bcrp1-positive subpopulation accounted for about 7% of the parent HeLa cell population. The proliferative capacity of the Bcrp1-positive cells was greater than that of the Bcrp1-negative cells (P < 0.05). In the invasion assay, the Bcrp1-positive cells demonstrated a greater invasive capacity through the artificial basement membrane than their Bcrp1-negative counterparts. Following transplantation of 10⁴ cells, only the Bcrp1-positive cells formed tumors in NOD/SCID mice. When 10⁵ or 10⁶ cells were transplanted, the tumor incidence and the tumor mass were greater in the Bcrp1-positive groups than those in the Bcrp1-negative groups (P < 0.05). The Bcrp1-positive subpopulation cervical cancer stem cells.     

Replication Fork Collapse and Genome Instability in a Deoxycytidylate Deaminase Mutant

Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1⁺ dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.     

The effect of the DNA-suppressing factor (DSF) on host DNA synthesis in synchronized cell cultures

Purified host DNA-suppressing factor (DSF) produced into culture fluid of HeLa C-9 cells infected with measles virus inhibited cellular DNA synthesis in HeLa cells. When purified DSF was added into cultures of synchronous HeLa cells at the early G1-phase, cellular DNA synthesis was irreversibly inhibited. However, DSF did not affect the stability of native double-stranded DNA nor the chain-elongation of single-stranded DNA in cells of the S-phase.     

Specific nucleotide sequences in HeLa cell inverted repeated DNA: enrichment for sequences found in double-stranded regions of heterogeneous nuclear RNA

Inverted repeat DNA was isolated from HeLa cell nuclei and transcribed in vitro with Escherichia coli RNA polymerase in the presence of [alpha-³²P]nucleoside triphosphates. The RNA products were digested with T₁ ribonuclease and subjected to separation in two dimensions. The pattern of the prominent oligonucleotides was almost indistinguishable from that seen when the double-stranded regions from ³²P-labeled HeLa cell heterogeneous nuclear RNA were fingerprinted in a similar manner. The sequences of several of the largest prominent T₁ ribonuclease-generated oligonucleotides were determined and were found to agree with those isolated from the double-stranded heterogeneous nuclear RNA that migrated to the same positions in the fingerprints. The most prominent component of the inverted repeat DNA appears to be sequences that are transcribed into double-stranded regions in heterogeneous nuclear RNA molecules.     

The effect of 313 nanometer light on initiation of replicons in mammalian cell DNA containing bromodeoxyuridine.

After unifilar substitution of thymine with bromouracil in either human HeLa or bovine cells, exposure to 313 nm light inhibits initiation of clusters of replicons. Since this treatment results in damage only to DNA and because the effect is the same as observed after 100-1000 rads of X-ray irradiation to the same cells, we infer that the effect of the later treatment is also mediated almost exclusively by DNA damage.     

Toxic effects of resazurin on cell cultures

Resazurin, introduced as a cell viability indicator under the trade name alamarBlue^®, is generally regarded as nontoxic when used according to manufacturer’s suggested shorter-term incubation time specifications. However, problems arise when exposure times are extended to longer-term cultures on the order of days. To assess the effect of resazurin over longer incubation times, MCF7 (HTB-22), MCF10A (CRL-10317), 3T3-L1 (CL-173), and D1 (CRL-12424) cultures were tested with varying amounts of resazurin over 4- and 8-day periods. MCF7, 3T3-L1, and D1 cells cultured for 8 days with 20 % alamarBlue^® had significantly less cell survivability. Specifically, levels of metabolic activity, deoxyribonucleic acid (DNA) concentration, and glucose consumption of the cell lines cultured for 8 days in medium with 20 % alamarBlue^® were significantly lower (p < 0.05) than metabolic activity, DNA concentration, and glucose consumption of MCF7 cells cultured for 8 days in medium with no alamarBlue^®. MCF7, 3T3-L1, and D1 cells used less glucose at concentrations as low as 5 %. Data also suggests the toxic effects are more pronounced in the cancerous cell line as compared to the noncancerous cells.     

Modification of X-Ray-Induced Killing of HeLa S3 Cells by Inhibitors of DNA Synthesis

After irradiation of HeLa S3 cells with 220 kv x-rays during G1, treatment with any of six inhibitors of DNA synthesis results in the progressive enhancement of cell killing (loss of colony-forming ability). Incubation with hydroxyurea, cytosine arabinoside, or hydroxylamine reduces survival five- to twentyfold in about 8 hr, following an x-ray dose of 400 rads. In contrast, treatment with 5-fluorodeoxyuridine, deoxyadenosine, or thymidine after this same dose reduces survival less than twofold during a comparable time interval. These differences occur at drug concentrations which reduce the rate of DNA synthesis by at least 95% (except in the case of hydroxylamine, which inhibits DNA synthesis to a smaller extent), but which kill no unirradiated cells during the treatment periods. When inhibition of DNA synthesis with either hydroxyurea or cytosine arabinoside is reversed by addition of appropriate precursors of DNA, the enhancement is abolished. With hydroxyurea, the rate of cell killing is dependent on the dose of x-rays previously administered, and the extent of enhancement seems to be related to the drug concentration. Imposition of a delay between irradiation and addition of hydroxyurea does not abolish the enhancement effect, but instead causes a proportional lag in its inception. Postirradiation treatment of S phase cells with either hydroxyurea or cytosine arabinoside also enhances killing. Furthermore, unlike early G1 cells, S cells (and, as shown previously, cells blocked at the G1-S transition) are sensitized by preirradiation exposure to hydroxyurea.     

Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells.

A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. In contrast, the pH optimum of the HeLa DNA primase was very sharp and fell between pH 7.9 and 8.2. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase and was eluted from single-stranded DNA agarose at higher salt concentrations than the host primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degrees C for several weeks, the DNA primase separated from the viral DNA polymerase. Separation or decoupling could also be achieved by gel filtration of the HSV polymerase:primase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, we believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA.     

A COMPARISON BETWEEN HETEROGENEOUS NUCLEAR RNA AND POLYSOMAL MESSENGER RNA IN HELA CELLS BY RNA-DNA HYBRIDIZATION

Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 µg of HeLa cell DNA binds 0.05–0.10 µg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.     

In Vivo Stoichiometry of Shelterin Components

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1·POT1 heterodimer was present 50–100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1·POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ∼10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1·POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.     

A DNA Polymerase-����Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

α-Accessory factor (AAF) stimulates the activity of DNA polymerase-α·primase, the only enzyme known to initiate DNA replication in eukaryotic cells (Goulian, M., Heard, C. J., and Grimm, S. L. (1990) J. Biol. Chem. 265 ,13221 -13230). We purified the AAF heterodimer composed of 44- and 132-kDa subunits from cultured cells and identified full-length cDNA clones using amino acid sequences from internal peptides. AAF-132 demonstrated no homologies to known proteins; AAF-44, however, is evolutionarily related to the 32-kDa subunit of replication protein A (RPA-32) and contains an oligonucleotide/oligosaccharide-binding (OB) fold domain similar to the OB fold domains of RPA involved in single-stranded DNA binding. Epitope-tagged versions of AAF-44 and -132 formed a complex in intact cells, and purified recombinant AAF-44 bound to single-stranded DNA and stimulated DNA primase activity only in the presence of AAF-132. Mutations in conserved residues within the OB fold of AAF-44 reduced DNA binding activity of the AAF-44·AAF-132 complex. Immunofluorescence staining of AAF-44 and AAF-132 in S phase-enriched HeLa cells demonstrated punctate nuclear staining, and AAF co-localized with proliferating cell nuclear antigen, a marker for replication foci containing DNA polymerase-α·primase and RPA. Small interfering RNA-mediated depletion of AAF-44 in tumor cell lines inhibited [methyl-³H]thymidine uptake into DNA but did not affect cell viability. We conclude that AAF shares structural and functional similarities with RPA-32 and regulates DNA replication, consistent with its ability to increase polymerase-α·primase template affinity and stimulate both DNA primase and polymerase-α activities in vitro.In eukaryotic cells, DNA replication is initiated at multiple origins internal to each chromosome; the origin recognition complex recruits cell division cycle and minichromosome maintenance proteins to form a preinitiation complex (1). At the G₁-S phase transition, the latter complex is activated by cyclin-dependent protein kinases leading to formation of an initiation complex that alters local DNA structure through DNA helicase activity (1, 2). The replication protein A (RPA)² is recruited to bind and stabilize single-stranded DNA (ssDNA) produced by the initiation complex (3, 4). RPA serves as an auxiliary factor for DNA polymerase-α (pol-α)·primase: it stabilizes the protein complex by direct interaction with both pol-α and primase subunits, and it reduces the misincorporation rate of pol-α, acting as a “fidelity clamp” (5, 6). The pol-α·primase complex consists of four subunits, including the catalytic pol-α subunit (p185), a regulatory B subunit (p70), and two primase subunits (p49 and p58). On an ssDNA template, the primase synthesizes short RNA primers from ribonucleoside triphosphates (rNTPs), which are elongated by pol-α in the presence of deoxyribonucleoside triphosphates (dNTPs) to form short DNA fragments. Through mechanisms requiring other replication factors, pol-α·primase is replaced by the more processive DNA polymerases pol-δ and pol-ε (7). Pol-ε synthesizes the leading strand, whereas pol-δ completes each Okazaki fragment initiated by pol-α·primase on the lagging strand and proofreads errors made by pol-α (7). The initiator RNA and DNA fragments are later removed by nucleases, and the Okazaki fragments are sealed by DNA ligase (7).The pol-α·primase complex is the only eukaryotic DNA polymerase able to initiate DNA synthesis de novo. In addition to initiating DNA replication and synthesizing Okazaki fragments, it appears to be one of the final targets of cell cycle checkpoint pathways that couple DNA replication to DNA damage response (2, 8). The role of RPA in initiation, elongation, and completion of lagging strand DNA synthesis has been thoroughly investigated (3, 9), but in vitro studies suggest that some additional factors that promote the rapidity of DNA replication in vivo are still lacking (2).In the course of purifying pol-α·primase from extracts of cultured mouse L1210 cells, we identified a factor we named α-accessory factor (AAF) that stimulates pol-α·primase activity in vitro (10, 11). The protein has a native molecular mass of ∼150 kDa as determined from its sedimentation coefficient and Stokes radius and is composed of two subunits of ∼132 and ∼44 kDa. AAF stimulates pol-α·primase activity with several different templates and types of reactions: (i) It stimulates selfprimed reactions with poly(dT), poly(dI·dT), or single-stranded circular DNA; (ii) it stimulates primed reactions with poly(dA)·oligo(dT) and multiply primed DNA in the absence of rNTPs, indicating that it affects pol-α activity when no primers are being made; and (iii) it stimulates primase activity on ssDNA in the absence of dNTPs, showing that it can enhance RNA primer synthesis in the absence of DNA synthesis (11). AAF increases the template affinity and processivity of pol-α·primase (12). AAF is highly specific for pol-α·primase and has no effect on the other mammalian DNA polymerases β, γ, or δ or on the DNA polymerase·primase complexes from Drosophila and Saccharomyces cerevisiae (11).The cloning of both AAF subunits based on peptide sequences obtained from the purified protein allowed us now to further characterize the AAF-44·AAF-132 complex structurally and functionally. Based on siRNA experiments in cancer cell lines, AAF appears to regulate DNA replication in vivo.     

p34(Cdc2) kinase activity is excluded from the nucleus during the radiation-induced G(2) arrest in HeLa cells

The progression of cells from G(2) into mitosis is blocked by exposure to DNA-damaging agents such as ionizing radiation. This G(2) delay is associated with reduced cyclin B1-specific associated histone H1 kinase activity, increased inhibitory phosphorylation of p34(Cdc2), and depressed cyclin B1 levels in HeLa cells. Induction of cyclin B1 or expression of Cdc2AF, a mutant p34(Cdc2) that lacks the sites of inhibitory phosphorylation, only partially reverses the radiation-associated G(2) delay, although both maneuvers rapidly result in increased histone H1 kinase activity. To account for the persistent G(2) delay in the face of active p34(Cdc2) kinase, we determined the location of the kinase activity. Although p34(Cdc2) was active in the cytoplasm, the nuclear p34(Cdc2) was inactive. Irradiation led to nuclear accumulation of the inactive tyrosine-phosphorylated form of p34(Cdc2), whereas the active form was seen in the cytoplasm. At later times when cells had resumed cell cycle progression, nuclear kinase activity was detectable. These results give evidence of segregation of cytoplasmic and nuclear kinase activity after DNA damage that has the effect of enhancing checkpoint control. Shielding the nucleus from the potentially deleterious effects of kinase activity after DNA damage may help irradiated human cancer cells respond to irradiation.