INCORPORATION OF TRITIATED THYMIDINE BY EUCARYOTIC MICROALGAE1

The uptake and incorporation of tritiated thymidine (³H-TdR) by axenic laboratory cultures of marine diatoms and dinoflagellates was measured. ³H-TdR was incorporated into nucleic acids by all four algae examined during a two to six hour period prior to cytokinesis and not during other times of the cell cycle. Between 90-95% of the ³H label incorporated into (cold trichloroacetic acid insoluble) nucleic acids was recovered from DNA. Incorporation of ³H-TdR appears to accurately indicate the timing of DNA synthesis. The incorporation of ³H-TdR by eucaryotic algae during long term (24 h) incubations does not generally preclude using ³H-TdR uptake to estimate bacterial production and growth during short term incubations.     

Influence of irradiation or thymidine (TdR) on the pattern of 3H-TdR incorporation at each cell position in the crypts of the small intestine of the mouse

Using autoradiographic methods it was noted that S phase cells at the bottom of the crypts in the small intestine were the most efficient scavengers of exogenous injected thymidine. The efficiency of the incorporation of 3H-TdR (salvage pathway of DNA synthesis) by cells at the crypt base (stem cell zone) was twice as high as for the S phase cells at the top of the crypt (maturing proliferative cells). There were no such position-dependent differences in incorporation of 3H-UdR (de novo pathway of DNA synthesis). Radiation (0.75-5.0 Gy 137Cs gamma-rays) inhibited the incorporation of 3H-TdR very rapidly and this was also cell-position dependent. The cells at the bottom of the crypt were the most affected. The injection of cold thymidine before 3H-TdR changed the pattern of the incorporation of 3H-TdR along the side of the crypt in a very similar way to radiation, and the grain number was decreased predominantly in the cells at lower positions. The possibility of the existence of a regional gradient of endogenous thymidine (reutilization from intestinal sources), and the influence of irradiation on the gradient of thymidine incorporation resulting from direct and abscopal effects of whole body exposure, are discussed.     

Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.     

Rates of incorporation of radioactive molecules during the cell cycle

We report measurements of the incorporation of radioactive molecules during short labeling periods, as a function of cell-cycle stage, using a cell-sorter-based technique that does not require cell synchronization. We have determined: (1) tritiated thymidine (³H-TdR) incorporation throughout S-phase in Lewis lung tumor cells in vitro both before and after treatment with cytosine arabinoside; (2) ³H-TdR incorporation throughout S-phase in KHT tumor cells in vitro and in vivo; (3) ³H-TdR incorporation throughout S-phase in Chinese hamster ovary cells and compared it with DNA synthesis throughout S-phase; (4) a mathematical expression describing ³H-TdR incorporation throughout S-phase in Chinese hamster M3-1 cells; and (5) the simultaneous incorporation of ³H-TdR and ³⁵S-methionine as they are related to cell size and DNA content in S49 mouse lymphoma cells. In asynchronously growing cells in vitro and in vivo, ³HH-TdR incorporation was generally low in early and late S-phase and highest in mid-S-phase. However, in Lewis lung tumor cells treated with cytosine arabinoside ³H-TdR incorporation was highest in early and late S-phase and lowest in mid-S-phase. Incorporation of ³⁵S-methionine increased continuously with cell size and DNA content. Incorporation of ³H-TdR in CHO cells was proportional to DNA synthesis.     

Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium-induced increase in thymic lymphoblast DNA synthesis

Increasing the extracellular calcium concentration in thymic lymphocyte suspension from 0.6 to 1.8 mM stimulated the proliferation of the lymphoblast subpopulation as measured by increases in the proportion of cells autoradiographically labeled with ³H-TdR and in mitotic activity. However it was not possible to show this increased DNA synthesis by scintillometric measurement of the amount of ³H-TdR incorporated into extracted DNA. On the other hand, calcium did raise the incorporation of ¹⁴C-formate into the thymine residues of DNA, and increased the activity of isolated thymocyte thymidylate synthetase. In contrast to the mitogenic calcium ion, a thymidylate synthetase inhibitor, methotrexate, actually increased the incorporation of ³H-TdR into DNA. It is concluded that calcium increases the endogenous synthesis of thymidylate which in turn prevents the amount of incorporation of exogenous ³H-TdR from accurately reflecting the true level of DNA synthesis.     

Repair of benzo[a]pyrene-initiated DNA damage in human cells requires activation of DNA polymerase alpha

Normal human fibroblasts treated with r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [3H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [3H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [3H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [3H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and 32P-ATP was introduced into the cells along with lipoproteins, 32P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.     

3H-thymidine incorporation method versus colony forming assay in the determination of anchorage-independent cell growth of rat sarcoma (XC) and Morris hepatoma 7777 (MH) cells.

The modification of 3H-thymidine incorporation method of Tanigawa was used to the estimation of anchorage-independent growth of virally and chemically transformed rat cells. The relationship between colony forming assay and 3H-TdR incorporation test was determined, depends on the composition of culture medium and the period of incubation of rat sarcoma (XC) cells with thymidine. The influence of exogenous mitogens (RFG, TGF beta 1 and insulin) and autocrine factor (at different step of purification) on the growth of Morris hepatoma 7777 (MH) cells was estimated by both methods. Regression analysis comparing the results of colony counting and thymidine incorporation revealed good correlation between the two methods. The modification can be used the determination of growth stimulating or growth inhibiting activity and in multistep purification procedure of autocrine growth factors.     

Thymidine metabolism and the monitoring of DNA synthesis in insects

The thymidine degradation pathway established for other organisms is confirmed in insects. When ³H-TdR is used as a marker for DNA synthesis in developing silkmoths, some is incorporated into DNA and some degraded to compounds not incorporated into DNA. After a single injection, ³H-TdR is rapidly cleared from haemolymph and other tissue, resulting in, at most, a 4 hr pulse. In wing tissue, detection of DNA synthesis is possible for a maximum of 4 hr after injection of precursor and for 6 hr in vitro. Continuous monitoring of DNA synthesis can be attained by perfusion, which maintains high levels of circulating ³H-TdR.     

Effects of removing PHA from cultures of PHA-stimulated lymphocytes

The proliferative capacity of PHA-stimulated lymphocytes following removal of PHA from the cultures was investigated. Lymphocytes were incubated with different PHA concentrations for 3 or 24 h and were then cultured in fresh medium with or without PHA in the original concentration. Cell proliferation was measured by incorporation of ³H-TdR. The effect of removing PHA was found to vary with the PHA concentration used for stimulation. Thus removal of PHA at 3 and 24 h from cells stimulated with half the optimal and at 3 h from cells stimulated with optimal PHA concentrations inhibited thymidine incorporation almost completely. Removal at 24 h from the latter cells resulted in a moderately decreased thymidine incorporation, whereas no decrease was seen after the removal of PHA from cells stimulated with twice the optimal concentration. When the cells were stimulated with very high PHA concentrations (20 × optimal), removal of PHA even resulted in an increased thymidine incorporation, a phenomenon that most probably has to do with the utilization of exogenous thymidine being inhibited by high PHA concentrations.The decreased thymidine incorporation after removal of low PHA concentrations was due to a reduction in the number of cells entering the proliferation cycle as well as to a decreased multiplication of cells already in DNA synthesis. This shows that PHA stimulates the cells even after they have initiated DNA synthesis. Various explanations for the results are discussed.     

Effects of Copper on Proliferation and Autocrine Secretion of Insulin-Like Growth Factor-1 (IGF-1) and IGF-Binding Protein-3 (IGFBP-3) in Chondrocytes from Newborn Pigs In Vitro

Chondrocytes from the lateral trochlear ridge of the distal femur taken from 1-day-old piglets were cultured in medium supplemented with 0, 7.8, 15.6, 31.2, and 62.5?μmol/L copper. Insulin-like growth factor-1 (IGF-1) and IGF-binding protein 3 (IGFBP-3) levels in culture medium were determined by radioimmunoassay. DNA synthesis in chondrocytes was measured by tritiated thymidine ((3)H-TdR) incorporation. Proliferation-promoting activity and incorporation of (3)H-TdR in chondrocytes were increased in all culture media supplemented with copper and 15% fetal calf serum (FCS). The contents of IGF-1 and IGFBP-3 were also enhanced significantly in culture media containing 15% FCS and supplemented with copper at 15.6, 31.2, and 62.5?μmol/L. The optimal copper concentration for promoting chondrocyte proliferation and autocrine secretion of IGF-1 and IGFBP-3 was 31.2?μmol/L.     

Diverse effects of three furocoumarins on human lymphocyte proliferation

The biological effects of three furocoumarins on the proliferation of human normal peripheral blood lymphocytes have been investigated. Mitogen-stimulated human lymphocytes were assayed "in vitro" by measuring 3H-thymidine (3H-TdR) incorporation in the presence and in the absence of 15-30 microM 3-carbethoxypsoralen (3-CPs), trimethylangelicin (TMA) and psoralen (PSR) with and without UV-A irradiation (365 nm). The three furocoumarins differ in their ability to form mono- and bi-functional adducts with DNA pyrimidine bases and in producing reactive species of oxygen. At low furocoumarin doses and short times of UV-A irradiation (15-30 sec) used in the present study, 3-CPs did not affect 3H-TdR incorporation in PHA-stimulated human lymphocytes, TMA strongly inhibited 3H-TdR incorporation, while, unexpectedly, PSR increased 3H-TdR incorporation in the absence of irradiation, likely acting, under these experimental conditions, as a co-mitogen.     

In vitro labeling of solid tissues with tritiated thymidine for autoradiographic detection of S-phase nuclei.

In vitro measurement of the thymidine labeling index (TLI) of solid tissues requires hyperbaric oxygenation and is potentiated by blockade of thymidylate synthetase by 5-fluoro-2'-deoxyuridine (FUdR) to favor uptake of tritiated thymidine (3H-TdR). Hyperbaric oxygenation can be achieved in a simple system through injection of oxygen into rubber-stoppered test tubes. Incubations are carried out in Hanks' balanced salt solution in a shaker bath at 37 C for 2 hours; an FUdR concentration of approximately 1 micron is optimal. Autoradiographic exposure for 1 week or less is sufficient for TLI measurements on human tissues. With 3 to 4 atmospheres oxygen tension, incorporation of 3H-TdR is sufficient for TLI measurement throughout slices of tissue cut 1 mm thick or less. Mincing of tissue is not necessary, and the anatomic continuity seen in ordinary histological preparations is preserved. A gradient of labeling intensity is present from the surface to the interior of the tissue, but sufficient intensity of labeling for detection of DNA synthesis can be achieved in the interior of the section. The gradient can be reduced only slightly by prior incubation in 3H-TdR with hyperbaric oxygen at 0 C. The method permits TLI measurements on the same specimens, including needle biopsies, that are used for pathologic diagnosis.     

转化细胞及腹水癌细胞对生长调节因子敏感性的研究

 本文~3H-TdR参入细胞DNA为指标研究了EGF等生长调节因子对小鼠腹水癌细胞DNA合成的影响,发现不同癌细胞对EGF等生长因子的敏感性有所差异,考虑到这也许与肿瘤细胞自身特性如恶性度有关。为了进一步探讨恶性度与这一敏感性是否相关,我们观察并比较了C_3H10T1/2CL_8(一种来源于鼠胚的正常成纤维细胞,简称NC_3H_(10)及转化的C_3H_(10)T1/2CL_8(用~3H-TdR转化的上述细胞,简称TC_3H_(10))对EGF等生长因子的敏感性。实验证明,细胞恶性转化后,对EGF的敏感性明显降低,~3H-TdR参入率降至原先的1/4以下。用DBcAMP作用于NC_3H_(10)和TC_3H_(10)均能抑制~3H-TdR参入DNA并可抑制EGF诱导的~3H-TdR参入作用。因此,我们认为,有关物理的致癌因素如放射性同位素,像生物、化学的致癌因素一样,亦能引起其转化细胞对外源性生长调节因子敏感性的改变。     

Changes in cellularity induced by radiation in a solid tumour.

The growth, and cellular responses of Morris hepatoma 3924 A to a locally-administered dose of 3750 R X-rays were studied using the following parameters; (1) relative tumour volume changes; (2) tritiated thymidine (3H-TdR) incorporation into DNA; (3) tumour DNA content and (4) cellular analysis, including 3H-TdR labelling index, mitotic index, aberrant mitotic frequency and relative cell density. Before depression of tumour growth, cell proliferation is temporarily interuppted. As proliferation is reinitiated, a short-lived synhcrony and prolongation of cell-cycle traverse are reflected in (a) the labelling index and mitotic index, (b) the relative cell density, and (c) the rate of incorporation of 3H-TdR into DNA. Within 4 days after radiation, cell proliferation and 3H-TdR incorporation are significantly depressed. Simultaneously there are reductions in both the relative cell density and tumour DNA contents, and these remain depressed as the tumours initiate regression. From these studies, it is apparent that the cellular responses to radiation insult occur well in advance of measurable volume changes and are observed both in tumours that continue to regress and in those that initiate regrowth.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

Kidney extract reversibly inhibits compensatory cell proliferation after unilateral nephrectomy

Unilateral nephrectomy (uNX) in mice is followed by a transitory increase in cell proliferation in the remaining kidney. To examine whether this response could be related to a negative feedback control of kidney epithelial cell renewal, water extracts were made of kidney homogenate. Five mg freeze-dried extract was injected 18 h post-operatively, and the animals were sacrificed at intervals during the following 54 h. The mitotic rate and the incorporation of tritiated thymidine (3H-TdR) into DNA were measured in the remaining kidney. The results show that the kidney extract reduces both the mitotic rate and the incorporation of 3H-TdR into DNA. In the tubular epithelium in the kidney, the strongest inhibitory effect was found by injecting the extract at 18 or 39 h postoperatively.     

Thymidine incorporation into algal DNA from axenic cultures of Synechococcus, Chlorella and Tetraselmis

The incorporation of tritiated ([³H]-) thymidine into DNA by axenic laboratory cultures of a common marine blue-green algae and two marine eukaryotic algae was measured. Tritiated thymidine was incorporated into the cold TCA fraction of the cyanobacterium and eukaryote algae. However, only in the culture of cyanobacterium was the thymidine consistently incorporated into DNA. Considering the usual algal densities in natural habitats, thymidine concentrations and incubation times, our data do not preclude the use of thymidine incorporation in bacterial production studies in marine environments.     

Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum)

The rate of [(3)H-methyl] thymidine ((3)H-TdR) incorporation into DNA has been applied extensively to measure cell production by bacterial communities in aquatic environments. Here we describe a method to quantify (3)H-TdR incorporation by specific, phylogenetically defined members of the bacterial community. The method involves selectively capturing DNA from targeted groups of bacteria and then quantifying its (3)H radioactivity. The method was applied to measure (3)H-TdR incorporation by the members of the phylum Bacteriodetes whose members, which include the Cytophaga-Flavobacter cluster, are ubiquitous in coastal waters. (3)H-labelled DNA from Bacteriodetes was selectively biotinylated in PCR-like reactions that contained a Bacteriodetes-specific 16S rRNA gene primer, thermostable DNA polymerase and biotinylated dUTP. The biotinylated DNA was then captured on streptavidin-coated beads and its (3)H radioactivity determined by scintillation counting. We have termed this method 'selective nucleic acid polymerase-biotinylation and capture' or 'SNAP-BAC'. Internal (33)P-labelled DNA standards were used to quantify the recovery of (3)H-labelled DNA from the SNAP-BAC reactions. The method was verified by successfully targeting Bacteriodetes in simple laboratory mixtures of (3)H-labelled DNA extracted from pure cultures of Bacteriodetes and gamma-proteobacteria. Field application of this method in Puget Sound and off the Washington coast determined that Bacteriodetes were responsible for 56 +/- 17% and 32 +/- 5% of community (3)H-TdR incorporation (1.3 +/- 0.3 and 9.9 +/- 1.7 pmol l(-1) h(-1)) at these two locations.     

Effect of Lithium on Schwann Cell Proliferation Stimulated by Axolemma- and Myelin-Enriched Fractions

Cultured Schwann cells stimulated with an axolemma- or myelin-enriched fraction incorporated 2.5 to three times as much [3H]thymidine when 10 mM lithium was added to the extracellular medium. The ability of lithium to enhance the mitogenic activity of either fraction was dose dependent. This result was not due to an increase in osmolarity, because addition of 10 mM NaCl had no effect on the amount of labeled thymidine accumulated by Schwann cells treated with either membrane fraction. In an earlier study, the effect of either membrane fraction could be potentiated with active phorbol esters. Lithium significantly enhanced the incorporation of [3H]thymidine into Schwann cells treated with a myelin-enriched fraction and phorbol esters. In contrast, lithium slightly increased the amount of labeled thymidine incorporated into Schwann cells stimulated with an axolemma-enriched fraction and phorbol esters. The mitogenic activity of either membrane fraction was impaired when the calcium channel blockers Mn2+ and nifedipine were added. Addition of lithium stimulated an increase in the amount of [3H]thymidine accumulated by Schwann cells treated with either the axolemma- or myelin-enriched fraction in the presence of either Mn2+ or nifedipine.     

Thymidine and Thymine Incorporation into Deoxyribonucleic Acid: Inhibition and Repression by Uridine of Thymidine Phosphorylase of Escherichia coli

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.