Dynamics of 3H-thymidine and 3H-deoxycytidine incorporation into the nuclei of a rabbit kidney cell culture during the S period

Using a combined cytophotometric-autoradiographic method a study was made of 3H-thymidine and 3H-deoxycytidine incorporation rates into the interphase nuclei of rabbit kidney cell culture during the S-period. The rate of 3H-deoxycytidine (10(-4) M--10(-6) M) incorporation into nuclei increases throughout the first part of the S-period and decreases from its middle to the end. The patterns of variations of 3H-thymidine and 3H-deoxycytidine incorporation rates into the nuclei of cultured rabbit kidney cells during the S-period were identical.     

Deoxycytidine is salvaged not only into DNA but also into phospholipid precursors. II. Ara-C does not inhibit the later process in lymphoid cells

dCTP formed from exogenous deoxycytidine via the salvage pathways was previously shown to serve deoxyliponucleotide synthesis in lymphocytes (Spasokukotskaja et al, Biochem. Biophys. Res. Commun. (1988) 155, 923-929) and now in lymphoma cells. After treatment with 1-beta-D-arabino-furanosylcytosine (ara-C), much more araCTP as well as araCDP-choline was formed in lymphoma cells than in lymphocytes explaining the high sensitivity of lymphoma cells to this drug. Ara-C did not inhibit labeling of 5-3H-dCDP-choline from exogenous 5-3H-deoxycytidine while inhibiting DNA synthesis. Excess of exogenous ribocytidine diminished labeling of araCDP-choline, without any effect on dCDP-choline. These data suggest that araCDP-choline and dCDP-choline were synthesized from separate pools in these cells.     

The effect of 5-azacytidine on the cell cycle and chromosome structure in cell cultures of swine embryonal kidney tissue

Incubation of pig kidney cells (PK-cells) in the presence of 5-azacytidine (5-azaC), a DNA enzymatic methylation inhibitor, at concentration of 20 microM for 6 or 24 h results in a dramatic decrease in the DNA methylation level (5mC/C + m5.100) - from 3.0 in control to 1.0 in experiment. This is accompanied by a virtually complete arrest of mitosis and a decrease in the ratio of labeled interphase cells upon simultaneous introduction of 3H-deoxycytidine. The incubation with 5-azaC block PK-cells mainly in the G2-period. The inhibitory action is reversible, for the cells enter into mitosis after removal of the inhibitor. Metaphase chromosomes, whose DNA was replicated in the presence of the 5-azaC, exhibit certain ultrastructural differences from normal ones. The results are being discussed in connection with the earlier data on the anomalous structure of interphase chromatin formed in the course undermethylated DNA replication.     

Characteristics of the metabolism and gonadal action in mice of tritium-labelled deoxycytidine

The levels of 3H-deoxycytidine binding in mice and rats were shown to depend on the amount of the carrier and the routes of administration, and the dynamics of its retention, on the dose and schedule of its administration. As estimated by the criterion of the testis mass decrease the 3H-deoxycytidine dose of 37 kBq/g is equivalent to gamma-radiation dose of 0.06 Gy. The coefficient of 3H-deoxycytidine absorption from the digestive tract of mice and rats is 2-10 times as high as that of 3H-thymidine, while both compounds administered to rats transcutaneously have the same absorption coefficients amounting to 0.02.     

The pattern of lymphocytes in the thymus and spleen after labeling with 3H-thymidine and 3H-deoxycytidine.

Adult male untreated mice (NMRI) were investigated after radioactive labeling with 3H-thymidine and 3H-deoxycytidine to find out whether the lymphocytes in the cortex and medulla of the thymus as well as in the perifollicular and periarteriolar regions of the spleen show a labeling pattern which allows a classification into T- and B-lymphocytes. The percentages of radioactively labeled small lymphocytes and their mean grain counts were determined. The percentages of radioactively labeled small lymphocytes after 3H-TdR and 3H-CdR showed no significant differences in both splenic zones. The grain counts over the lymphocyte nuclei in the periarteriolar zone showed lower values after 3H-TdR than after 3H-CdR. The lymphocytes in the perifollicular zone were strongly labeled with 3H-TdR and weakly labeled with 3H-CdR. In the thymus medulla, lymphocytes were weakly labeled with 3H-thymidine and strongly labeled with 3H-CdR. In the cortex no significant differences were observed. 75 to 80% of the small lymphocytes in the peripheral blood were weakly and 20-25% strongly labeled after 3H-TdR. Therefore there are similarities in the radioactive labeling pattern of thymic medulla lymphocytes and that of small lymphocytes of the periarteriolar zone of the spleen by both DNA precursors. The small lymphocytes in the peripheral T-dependent tissue zones, for example in the spleen, as well as in the mixed lymphocyte population of the peripheral blood can be differentiated from the B-lymphocytes through the difference in the amount of incorporation of 3H-thymidine and 3H-deoxycytidine.     

Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA

Methylation of deoxycytidine incorporated by DNA excision-repair was studied in human diploid fibroblasts following damage with ultraviolet radiation, N-methyl-N-nitrosourea, or N-acetoxy-2-acetylami-nofluorene. In confluent, nondividing cells, methylation in repair patches induced by all three agents is slow and incomplete. Whereas after DNA replication in logarithmic-phase cultures a steady state level of 3.4% 5-methylcytosine is reached in less than 2 hr after cells are labeled with 6-³H-deoxycytidine, following ultraviolet-stimulated repair synthesis in confluent cells it takes about 3 days to reach a level of ～2.0% 5-methylcytosine in the repair patch. In cells from cultures in logarithmic-phase growth, 5-methylcytosine formation in ultraviolet-induced repair patches occurs faster and to a greater extent, reaching a level of ～2.7% in 10–20 hr. Preexisting hypomethylated repair patches in confluent cells are methylated further when the cells are stimulated to divide; however, the repair patch may still not be fully methylated before cell division occurs. Thus DNA damage and repair may lead to heritable loss of methylation at some sites.     

Comparative study of human chromosome replication in primary cultures of embryonic fibroblasts and in cultures of peripheral blood leucocytes

Distributions of AT- and GC-base pairs along the length of chromosomes 1, 2, 3 and 16 in primary cultures of embryonic fibroblasts and of peripheral blood leucocytes were studied by autoradiography with: 1. ³H-thymidine and ³H-deoxycytidine; 2. ³H-deoxyadenosine and ³H-deoxyguanosine. It has been shown that the two types of cells differ in the DNA content and proportion of AT- and GC-nucleotide pairs in the centromeric heterochromatin of chromosome I: this region contains more DNA in fibroblasts than in leucocytes mainly due to AT-pairs. In both types of cells the telomeric region of the short arm of this chromosome contains more GC- than AT-pairs. Similar results were obtained for C-heterochromatin of chromosome 16: the frequencies of labelling of this region by ³H-deoxyadenosine and ³H-thymidine in fibroblast cultures were higher than in case of ³H-deoxycytidine and ³H-deoxygyanosine, and in leucocyte cultures these frequencies were almost equal. No differences in the distributions of base pairs along the length of chromosome 2 and 3 were established in the two types of cells. — The nature of the established phenomenon may be connected with under-replication or loss in another way of part of the genetic material in the process of development and differentiation of cell systems.     

A non-radioactive sensitive assay to measure 5-fluorouracil incorporation into DNA of solid tumors

A non-radioactive method to determine 5-Fluorouracil (5FU) incorporation into DNA has been developed. Isolated DNA was enzymatically degraded to bases and the resulting 5FU was measured with standard gas-chromatography coupled to mass spectrometry (GC-MS) and compared with that of radioactive 5FU in a cell line. Incorporation into DNA of the murine Colon 26-B tumor treated with maximal tolerated doses of 5FU and fluorodeoxyuridine (FUdR) was maximal after 2 hour and was 15.4 and 71.0 fmol/microg DNA, respectively. After a plateau for about 3 days a decrease was observed to +/- 2 fmol/microg DNA after 10 days. The assay is very sensitive and reproducible and can be used in a clinical setting.     

Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors

DNA fibre autoradiography, after incorporation of high specific activity 3H-thymidine and 3H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) or G1 (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however, without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G1 cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.     

Incorporation of 14C-thymidine into liver and brain DNA of protein-deficient rats.

The authors studied the effect of a protein-free diet on 14C-thymidine incorporation into rat liver DNA in vivo and found, after 2-3 weeks, a marked decrease in uptake of the radioactive base into the liver DNA, followed by a decrease in the proportion of DNA in liver cell homogenates. The total nuclear count/mg liver tissue displayed an increase during protein depletion, except for the 5th week, when a decrease was recorded. The incorporation of 14C thymidine into the brain DNA likewise displayed no great differences, although a significant drop was observed during the 2nd to 4th week of depletion. In the 5th week we recorded an increase in uptake of the radioactive base by brain DNA, exceeding the incorporation values in the controls.     

Mutagenic nucleoside analog N4-aminocytidine: metabolism, incorporation into DNA, and mutagenesis in Escherichia coli.

N4-Aminocytidine, a nucleoside analog, is strongly mutagenic to various organisms including Escherichia coli. Using E. coli WP2 (trp), we measured the incorporation of [5-3H]N4-aminocytidine into DNA and at the same time measured the frequency of reversion of the wild type, thereby attempting to correlate the incorporation with mutation induction. First, we observed that N4-aminocytidine uptake by the E. coli cells was as efficient as cytidine uptake. High-pressure liquid chromatographic analysis of nucleoside mixtures obtained by enzymatic digestion of isolated cellular DNA showed that the DNA contained [3H]N4-aminodeoxycytidine, corresponding to 0.01 to 0.07% of the total nucleoside; the content was dependent on the dose of N4-aminocytidine. There was a linear relationship between the N4-aminocytosine content in DNA and the mutation frequency observed. These results constitute strong evidence for the view that the N4-aminocytidine-induced mutation in E. coli is caused by the incorporation of this agent into DNA as N4-aminodeoxycytidine. We also found that the major portion of radioactivity in DNA of cells that had been treated with [5-3H]N4-aminocytidine was in the deoxycytidine fraction. We propose a metabolic pathway for N4-aminocytidine in cells of E. coli. This pathway involves the formation of both N4-aminodeoxycytidine 5'-triphosphate and deoxycytidine 5'-triphosphate; the deoxycytidine 5'-triphosphate formation is initiated by conversion of N4-aminocytidine into uridine. In support of this proposed scheme, a cytidine deaminase preparation obtained from E. coli catalyzed the decomposition of N4-aminocytidine into uridine and hydrazine.     

Effects of FSH and LH on incorporation of [3H]thymidine into follicular DNA

To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1-4 correspond to follicles with 1-4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7-8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8-10 = small, intermediate and large antral follicles, respectively. Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1-9. The optimal treatment to reverse the effects of phenobarbitone was 1 microgram FSH and 2 micrograms LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2-10: FSH acted primarily on Stages 2-5 and LH on Stages 5-10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2.5 micrograms FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1-8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2.5 micrograms FSH restored DNA synthesis 6 h later to Stage 2-6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Method for Restricting Incorporation of Radioactivity from 3H-Thymidine into Deoxyribonucleic Acid Only During Outgrowth of Spores of Bacillus cereus T

When heat-activated spores of Bacillus cereus T (thy(-)) were germinated and grown in medium containing (3)H-thymidine, a significant amount of radioactivity was incorporated into ribonucleic acid and deoxyribonucleic acid (DNA). A method was developed to restrict the incorporation of radioactivity from (3)H-thymidine into DNA only. This was accomplished by labeling the cells with (3)H-thymidine in the presence of 2 mg of 2-deoxyadenosine per ml, 250 mug each of uracil, cytosine, and guanosine per ml, and 500 mug of adenosine per ml. Under these conditions, 97% of the radioactivity incorporated into cold trichloroacetic acid-insoluble material was associated with DNA only. In the absence of these compounds, DNA contained only 72% of the total radioactivity incorporated into cold acid-insoluble material.     

Incorporation of 2'-deoxy-5-(trifluoromethyl)uridine and 5-cyano-2'-deoxyuridine into DNA.

In an attempt to synthesize DNA containing 2'-deoxy-5-(trifluoromethyl)uridine (1) using previously published protocols, we found that the trifluoromethyl group converted into a cyano group, resulting in DNA containing 5-cyano-2'-deoxyuridine (3). We show that nucleoside 1 can be incorporated into DNA using phosphoramidite 2 in combination with acetyl-protected deoxycytidine and phenoxyacetyl-protected purine phosphoramidites. Replacing thymidine in DNA with 1 caused a slight decrease in DNA duplex stability at pH 6.9.     

The uptake of [3H]uridine into the nucleic acids of rat uterus during early pregnancy

1. Changes in content and uptake of [(3)H]uridine into the nucleic acids of rat uterus during the first 9 days of pregnancy were studied. 2. From day 6 implantation sites were separated from the rest of the uterine tissue for independent analysis. 3. Up to day 5 of pregnancy no changes were found in the total dry matter or in RNA and DNA content/unit dry matter nor in the RNA/DNA ratios. 4. From day 6, when implantation sites are visible, the water content of the implantation sites increased by 2-3%, and the RNA content/unit dry wt. and the RNA/DNA ratios increased. The DNA content/unit dry wt. did not increase in the implantation sites until day 8. 5. Uptake of [(3)H]uridine into the acid-soluble fraction of the tissues was markedly higher in implantation sites than in non-implantation sites. 6. Uptake of [(3)H]uridine into RNA was significantly increased on day 3 of pregnancy and again on day 5. 7. On days 6 and 7, the incorporation into RNA of implantation sites was significantly higher than in the remainder of the uterine tissue but decreased on days 8 and 9 to the same value as that of the normal tissue. 8. No change occurred in uptake into DNA until day 6, when there was an increase in uptake by the implantation sites. 9. It is suggested that the increase in RNA synthesis on day 3 is a preparation of the uterus for the onset of implantation on day 5, and that increased synthesis in implantation sites on days 6 and 7 is the elaboration of new RNA necessary for this early stage of pregnancy to commence.     

On the impossibility of the incorporation of 5-methylcytosine and its nucleosides into higher plant DNA

No radioactivity was detected in 5-methylcytosine isolated from wheat DNA after incubation of wheat seedlings with 3H-labelled 5-methylcytosine, 5-methylcytidine and 5-methyldeoxycytidine. No label from 3H-5-methylcytosine was found in DNA of seedlings. After incubation of seedlings with 3H-labelled nucleosides of 5-methylcytosine, radioactivity was discovered only in thymine of DNA. Thus 5-methylcytosine and its nucleosides can not be used in plants as direct precursors of 5-methyl cytosine residues in DNA, but nucleosides of 5-methylcytosine may be deaminated to thymidine (or deoxythymidine) and subsequently incorporated into DNA.     

The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10(-9)-5 X 10(-7) M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10(-4) M. However, at the highest FdUrd dose tested (10(-7) M), 10(-4) M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6-3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6-3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2'-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

Interstitial telomeres are hotspots for illegitimate recombination with DNA molecules injected into the macronucleus of Paramecium primaurelia.

DNA molecules injected into the macronucleus of Paramecium primaurelia replicate either as free linear telomerized or chromosome integrated molecules. In the present study we show that when a 1.77 kb BamHI DNA fragment harbouring the his3 gene of Saccharomyces cerevisiae was microinjected into the macronucleus, a fraction of the molecules are integrated into the chromosome via an illegitimate recombination process. The injected molecules were mostly inserted at their extremities at multiple points in the genome by replacing the Paramecium sequences. However, insertion sites were not totally at random. Roughly 30% of the molecules were integrated next to or in telomeric repeats. These telomeric repeats were not at the extremities of chromosomes but occupy an internal or interstitial position. We argue that such sites are hotspots for integration as the probability of random insertion near or in an interstitial telomeric site, of which there are 25-60 in a macronucleus is between 5 x 10(-4) and 3 x 10(-5).     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.