Hydroxyurea: effects on deoxyribonucleotide pool sizes correlated with effects on DNA repair in mammalian cells

We have measured deoxyribonucleotide pool sizes in different cell types: normal human, transformed human (HeLa), and the permanent hamster line CHO-K1. The range of sizes of the four DNA precursor pools in CHO cells is far greater than in human cells. It is a general rule that hydroxyurea causes rapid depletion of pools (except for dTTP) until the pool present in smallest amount is exhausted; this suggests a tight coupling of the pools to DNA replication (the presumed main cause of the depletion). The effect of hydroxyurea on DNA repair after ultraviolet irradiation (namely, a relatively small accumulation of incomplete repair sites blocked at the resynthesis stage) is probably accounted for by the reduced availability of DNA precursors. However, depletion of the dCTP pool is not an adequate explanation for the observed enhancement by hydroxyurea of the inhibitory effect of cytosine arabinoside; we suggest other possible modes of action. Ultraviolet irradiation has only small effects on the levels of deoxyribonucleotides.     

Cell cycle-dependent variations in deoxyribonucleotide metabolism among Chinese hamster cell lines bearing the Thy- mutator phenotype.

Deoxyribonucleotide pool imbalances are frequently mutagenic. We have studied two Chinese hamster ovary cell lines, Thy- 49 and Thy- 303, that were originally characterized by M. Meuth (Mol. Cell. Biol. 1:652-660, 1981). In comparison with wild-type CHO cells, both lines have elevated dCTP/dTTP ratios, resulting from loss of feedback control of CTP synthetase. While asynchronous cultures of both cell lines contain nearly identical deoxyribonucleoside triphosphate (dNTP) pools and both display elevated spontaneous mutation frequencies, the mutation frequencies between the two cell lines differ by as much as 10-fold. We asked whether differences in dNTP pools could be seen in extracts of rapidly isolated nuclei. Small differences, probably not large enough to account for the differences in mutation frequencies, were seen. However, when synchronized S-phase-enriched cell populations were examined, substantial differences were seen, both in whole-cell extracts and in nuclear extracts. Thy- 303 cells, which have higher mutation frequencies than do Thy- 49 cells, also showed the more aberrant dNTP pools. These data indicate that the Thy- 303 line contains a second mutation in addition to the mutation affecting CTP synthetase control. Evidence suggests that this putative second mutation affects an allosteric regulatory site of ribonucleotide reductase. The data on intranuclear dNTP pools in synchronized S-phase cells indicate that higher proportions of cellular dATP and dGTP are found in the nucleus than are corresponding amounts of dCTP and dGTP. Thus, despite the porous nature of the nuclear membrane, there are conditions under which the distributions of deoxyribonucleotides across this membrane are not random.     

Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.

The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools.     

Mode of orthophosphate uptake and ATP labeling by mammalian cells.

Incubation of HeLa cells with [32P]orthophosphate results in more rapid labeling of the gamma-phosphorus of ATP than of the intracellular pool of orthophosphate. The specific radioactivity of ATP equals that of extracellular orthophosphate after 2 h of incubation. A similar pattern of labeling is seen with human erythrocytes when incubated at physiological concentrations of orthophosphate (2 mM) and pH 7.4-7.8. At lower pH, 6.8-7.2, the rate of orthophosphate uptake increases and exceeds the rate of labeling of ATP. These data are explained by the existence of a primary system for ATP uptake which involves the mediation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. Phosphate first enters the cell as 1,3-diphosphoglyceric acid, is then transferred to ATP, and then enters the intracellular orthophosphate pool. At lower pH monovalent orthophosphate also enters the erythrocyte by a process not involving glyceraldehyde-3-phosphate dehydrogenase.     

DNA labeling in living cells.

BACKGROUND: Live cell fluorescence microscopy experiments often require visualization of the nucleus and the chromatin to determine the nuclear morphology or the localization of nuclear compartments. METHODS: We compared five different DNA dyes, TOPRO-3, TOTO-3, propidium iodide, Hoechst 33258, and DRAQ5, to test their usefulness in live cell experiments with continuous imaging and photobleaching in widefield epifluorescence and confocal laser scanning microscopy. In addition, we compared the DNA stainings with fluorescent histones as an independent fluorescent label to mark chromatin. RESULTS: From the dyes tested, only Hoechst and DRAQ5 could be used to stain DNA in living cells. However, DRAQ5 had several advantages, namely low photobleaching, labeling of the chromatin compartments comparable to that of H2B-GFP fusion proteins, and deep red excitation/emission compatible with available genetically encoded fluorescent proteins such as C/G/YFP or mRFP. CONCLUSIONS: The DNA dye DRAQ5 is well suited for chromatin visualization in living cells and can easily be combined with other fluorophores with blue to orange emission.     

Biosynthetic labeling of hypusine in mammalian cells. Carbon-hydrogen bond fissions revealed by dual labeling

Using a dual-label technique in which 3H- and 14C-labeled forms of putrescine and of spermidine were employed as biosynthetic precursors of hypusine, two -C-H bond cleavages were detected during production of this unique amino acid in Chinese hamster ovary cells. One of these cleavages occurs at C-1 of the 4-aminobutyl group during its transfer from the secondary amine nitrogen of spermidine to the nitrogen at the epsilon-position of a specific lysine residue in the polypeptide precursor of eukaryotic initiation factor 4D. Breakage of the other -C-H bond takes place at C-2 in this aminobutyl segment after it has been coupled to lysine to form the intermediate deoxyhypusine residue. Hydroxylation at this carbon atom, which constitutes the last step in hypusine biosynthesis, is the cause of bond cleavage. The data obtained are consistent with a notion that no additional -C-H bond fissions occur during hypusine biosynthesis. Our findings permit suggestion of a mechanism for enzymic aminobutyl group transfer in which 4-aminobutyraldehyde produced by oxidative cleavage of spermidine is coupled with the epsilon-amino group of a specific lysine residue to form an enzyme-bound imine intermediate.     

Cell killing and DNA damage induced in cultured mammalian cells by some tetrahydrobenzopsoralenquinones

The mechanism of action of two tetrahydrobenzopsoralenquinones: 4-methyl-tetrahydrobenzopsoralenquinone (compound 3) and 4-hydroxymethyltetrahydrobenzopsoralenquinone (compound 4) was studied in mammalian cells. These agents differ structurally from earlier benzo and tetrahydrobenzopsoralen derivatives 4-hydroxymethylbenzopsoralen (compound 1) and 4-hydroxymethyltetrahydrobenzopsoralen (compound 2) by the replacement of the benzopyranone with a quinonepyranone. In this study, we evaluated the antiproliferative activity of such derivatives in normal human lymphocytes and CHO cells cultivated in vitro. Compound 4 showed a noticeable antiproliferative activity. Studying the induction of chromosomal aberrations and of SCEs, we demonstrated that compound 4 has a clastogenic effect on mammalian cells. By means of DNA filter elution and protein precipitation techniques we evaluated the DNA damage produced by the tested compounds. Some experiments performed in presence of a DNA synthesis inhibitor showed that ongoing DNA synthesis is involved in cell killing by derivative 4. All data obtained suggest that compound 4 can interfere with the activity of topoisomerase II. Catalytic studies carried out with purified topoisomerase II and bacteriophage DNA confirmed this hypothesis.     

Cell cycle-dependent effects of wortmannin on radiation survival and mutation

Wortmannin, a known radiation sensitizer, has been used in experiments with synchronized cells to compare its effect on radiation survival and mutation induction within the cell cycle. PL61 cells (CHO cells with an inactivated HPRT gene containing a single active copy of a bacterial gpt gene) were synchronized by mitotic selection. Wortmannin administered before gamma irradiation caused a greater sensitization in G(1)-phase cells relative to late S/G(2)-phase cells. Preferential radiosensitization of G(1)-phase cells by wortmannin sets a limit to the proposed use of wortmannin in radiation therapy, since, in contrast to normal tissues, tumors usually have high proportions of S-phase cells. Wortmannin increased mutation frequencies in both G(1)- and S/G(2)-phase cells. Interestingly, relative increases in radiation-induced mutations in G(1) and S/G(2) phases were comparable. The results are discussed in terms of the contributions of different repair modes in the production of mutations.     

Mode of orthophosphate uptake and ATP labeling by mammalian cells

Incubation of HeLa cells with [³²P]orthophosphate results in more rapid labeling of the γ-phosphorus of ATP than of the intracellular pool of orthophosphate. The specific radioactivity of ATP equals that of extracellular orthophosphate after 2h of incubation. A similar pattern of labeling is seen with human erythrocytes when incubated at physiological concentrations of orthophosphate (2 mM) and pH 7.4–7.8. At lower pH, 6.8–7.2, the rate of orthophosphate uptake increases and exceeds the rate of labeling of ATP. These data are explained by the existence of a primary system for ATP uptake which involves the mediation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. Phosphate first enters the cell as 1,3-diphosphoglyceric acid, is then transferred to ATP, and then enters the intracellular orthophosphate pool. At lower pH monovalent orthophosphate also enters the erythrocyte by a process not involving glyceraldehyde-3-phosphate dehydrogenase.     

Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits

Consistent with its specialized role in DNA synthesis, the activity of ribonucleotide reductase is cell cycle-dependent, reaching its maximum during S-phase. This paper demonstrates, however, the levels of the two protein subunits, M1 and M2, of this enzyme vary independently of one another. The level of protein M1 was determined by use of a two-site monoclonal antibody-enzyme immunoassay and found to be constant throughout the cell cycle in bovine kidney MDBK cells. Pulse-chase experiments showed that the half-life of protein M1 was 15 h. This contrasts with our previous results demonstrating an S-phase-correlated increase in the concentration of protein M2 and a half-life of this subunit of 3 h. Therefore, ribonucleotide reductase is controlled during the cell cycle by the level of protein M2.     

Altered deoxyribonucleotide pools in T-lymphoblastoid cells expressing the multisubstrate nucleoside kinase of Drosophila melanogaster

The multisubstrate nucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human solid tumor cells and its unique enzymatic properties makes this enzyme a suicide gene candidate. In the present study, Dm-dNK was stably expressed in the CCRF-CEM and H9 T-lymphoblastoid cell lines. The expressed enzyme was localized to the cell nucleus and the enzyme retained its activity. The Dm-dNK overexpressing cells showed approximately 200-fold increased sensitivity to the cytostatic activity of several nucleoside analogs, such as the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 1-beta-d-arabinofuranosylthymine (araT), but not to the antiherpetic purine nucleoside analogs ganciclovir, acyclovir and penciclovir, which may allow this technology to be applied in donor T cells and/or rescue graft vs. host disease to permit modulation of alloreactivity after transplantation. The most pronounced effect on the steady-state dNTP levels was a two- to 10-fold increased dTTP pool in Dm-dNK expressing cells that were grown in the presence of 1 microm of each natural deoxyribonucleoside. Although the Dm-dNK expressing cells demonstrated dNTP pool imbalances, no mitochondrial DNA deletions or altered mitochondrial DNA levels were detected in the H9 Dm-dNK expressing cells.     

Altered nucleoside transporters in mammalian cells selected for resistance to the physiological effects of inhibitors of nucleoside transport

From a mutagenized population of wild type S49 T lymphoma cells, clones were generated that were resistant to the physiological effects of the potent inhibitor of nucleoside transport, 4-nitrobenzyl-6-thioinosine (NBMPR). These cells were selected for their ability to survive in semisolid medium containing 0.5 mM hypoxanthine, 0.4 microM methotrexate, 30 microM thymidine, 30 microM deoxycytidine, in the presence of 30 microM NBMPR. NBMPR protected wild type cells from the effects of a spectrum of cytotoxic nucleosides, whereas two mutant clones, KAB1 and KAB5, were still sensitive to nucleoside-mediated cytotoxicity in the presence of NBMPR. Comparisons of the abilities of wild type cells and mutant cells to incorporate exogenous nucleoside to the corresponding nucleoside triphosphate indicated that the KAB1 and KAB5 mutant cells were refractory to normal inhibition by NBMPR. Moreover, rapid transport studies indicated that mutant cells, unlike wild type parental cells, had acquired a substantial NBMPR-insensitive nucleoside transport component. Binding studies with [3H]NBMPR indicated that KAB5 cells were 70-75% deficient in the number of NBMPR binding sites, whereas KAB1 cells possessed a wild type complement of NBMPR binding sites. These data suggest that the NBMPR binding site in wild type S49 cells is genetically distinguishable from the nucleoside carrier site.     

Variable effects of DNA-synthesis inhibitors upon DNA methylation in mammalian cells.

Post-synthetic enzymatic hypermethylation of DNA was induced in hamster fibrosarcoma cells by the DNA synthesis inhibitors cytosine arabinoside, hydroxyurea and aphidicolin. This effect required direct inhibition of DNA polymerase alpha or reduction in deoxynucleotide pools and was not specific to a single cell type. At equivalently reduced levels of DNA synthesis, neither cycloheximide, actinomycin D nor serum deprivation affected DNA methylation in this way. The topoisomerase inhibitors nalidixic acid and novobiocin caused significant hypomethylation indicating that increased 5-mCyt content was not a necessary consequence of DNA synthesis inhibition. The induced hypermethylation occurred predominantly in that fraction of the DNA synthesized in the presence of inhibitor; was stable in the absence of drug; was most prominent in low molecular weight DNA representing sites of initiated but incomplete DNA synthesis; and occurred primarily within CpG dinucleotides, although other dinucleotides were overmethylated as well. Drug-induced CpG hypermethylation may be capable of silencing genes, an effect which may be relevant to the aberrantly expressed genes characteristic of neoplastic cells.     

Genetic effects of specific DNA lesions in mammalian cells

Accurate dosimetry for chemical mutagens is extremely difficult, and precise manipulation of the frequency of a particular lesion is ordinarily impossible. With 8-MOP plus UVA, however, both are possible because 8-MOP, when photoactivated by one photon of UVA, forms monoadducts whilst crosslinks are formed only if a second photon of light photoactivates the monoadducts. If 8-MOP molecules that are unreacted after a UVA exposure are removed from cells by washing, the effect of a subsequent UVA irradiation can be attributed only to the conversion of monoadducts to DNA interstrand crosslinks. Using this experimental procedure and L5178Y mouse lymphoma cells, we have shown that DNA interstrand crosslinks are at least 10-fold more effective at causing both sister-chromatid exchanges and chromosomal aberrations than are monoadducts. In contrast, crosslinks are no more effective than monoadducts in mutation induction. These experiments identify directly for the first time that a particular chemically induced lesion, DNA interstrand crosslinks, can, like thymine dimers, cause chromosomal aberrations and sister-chromatid exchanges. The results also show that sister-chromatid exchanges can be induced independently of mutations.