Rate of DNA synthesis

Human tissues can synthesize purines and pyrimidines from amphibolic intermediates. Ingested nucleic acids and nucleotides, which therefore are dietarily nonessential, are degraded in the intestinal tract to mononucleotides, which may be absorbed or converted to purine and pyrimidine bases. The purine bases are then oxidized to uric acid, which may be absorbed and excreted in the urine. While little or no dietary purine or pyrimidine is incorporated into tissue nucleic acids, injected compounds are incorporated. The incorporation of injected [ H] thymidine into newly synthesized DNA thus is used to measure the rate of DNA synthesis. 

Aminouracil produces a block in the mitotic cycle of various plants. For example, cessation of mitosis occurred in Vida faba roots incubated 24 h with this compound [392—394]. Depending on different experimental conditions, thymidine or thymidylic acid may or may not alleviate these effects [392—394]. It was concluded that 5-aminouracil depressed the rate of DNA synthesis, which led to an accumulation of cells in the S phase. After removal of the agent, DNA synthesis resumed. Similar results have been observed with Allium cepa and Haplopappus gracilis [395, 396]. Inhibition of guanosine incorporation into RNA of meristematic cells in Vida faba by 5-aminouracil was also reported [397]. 

The rate of DNA synthesis in a culture of cells could be most accurately determined by measuring the incorporation of which of the following radioactive compounds ... 

Correct answer = D. Because thymidine is essentially found only ii DNA, its incorporation wodd most accurately reflect the rate of DNA synthesis. Uridine is found only in RNA and could be used to measure the rate of RNA synthesis. Phosphate, adenine, and guanine are present in both DNA and RNA, and could not be used to speciflcaly measure synthesis of either one. 

If cells are synchronised at the G1 /S boundary and then released, the rate of DNA synthesis is initially slow but accelerates to reach a maximum at about 3h and then decelerates until S-phase is essentially complete in 6-7 h (Stubblefield and Mueller, 1962 Adams, 1969b). As replication occurs different numbers of replicons are active at any one time, and so it is not surprising that more careful labelling reveals bursts of tritiated thymidine incorporation throughout S-phase rather than a steady even progression (Klevecz, 1969 Lett and Sun, 1970 Klevecz et al., 1974). 

The requirements for epithelial cells are somewhat different (Reiss and Dibble, 1988). Mouse keratinocytes (MK-1 cells) enter a GO-phase within 24 h when confluent cultures are fed a serum-free, low Ca2+ (< 0.1 mM) medium supplemented with insulin, transferrin and sodium selenate (see 5.8). Addition of EGF (10 ng/ml) causes cells to enter S-phase after 10-12 h although the percentage of cells responding is not known. Insulin is not essential for this effect but apparently leads to a threefold increase on the rate of DNA synthesis measured 22-24 h after addition of EGF. TGF/ (100 pM) completely abolishes the effect of EGF. 

Thymidine is taken up by cells and rapidly converted to dTTP, the pool size of which is related to the extracellular thymidine concentration (see 12.1). At thymidine concentrations as low as 3 x 10-7M this leads to a measurable effect on the rate of DNA synthesis (Cooper et al., 1966). At concentrations above 1 mM inhibition of DNA synthesis is almost complete for some cell lines (Morris and Fischer, 1960 Xeros, 1962 Bootsma et al., 1964 ... 

Estimates of the rate of DNA synthesis in a cell population or in individual cells may be required as a measure of the rate of cell growth, or for cell cycle studies, or to satisfy a basic interest in DNA metabolism. However, on addition of tritiated thymidine to cells in culture a number of problems arise. 

Should the cell have a permeability barrier or should the various kinases be rate-limiting, then the incorporation would not reflect the rate of DNA synthesis. Rather, incorporation would be dependent on the intermediate, rate-limiting enzyme. 

The endogenously synthesised dTTP dilutes the specific activity of the [3H]dTTP formed from the added [3H]thymidine. Thus on adding tritiated thymidine at 3 X 10 8M most of the DNA thymine is synthesised by the endogenous or de novo pathway, but when the [3H]thymidine concentration in the medium is raised to 0.3 mM it contributes 90% or more of the DNA thymine (Cleaver and Holford, 1965 Cooper et al., 1966 Cleaver, 1967). As the specific activity of [3H]dTTP is one of the factors which determine the amount of radioactivity incorporated into DNA (either total counts/min or grain counts) and as this varies (a) with external thymidine concentration and (b) with the state of the cells, the quantitative estimation of rates of DNA synthesis is full of pitfalls. 

The rate of incorporation of radioactivity is dependent on the specific activity of the [3H]dTTP which in the two previous methods has been assumed to be the same as that of the supplied [3H]thymi-dine. As the concentration of [3H] thymidine is increased from low values (i.e. less than 10-6M) the specific activity of the [3H]dTPP pool rises and so does the incorporation of radioactivity into DNA. This is at a time when the true rate of DNA synthesis remains unchanged. Thus the proporation of the dTTP pool which is radioactive, ([3H]dTTP(dTTP + [3H]dTTP)), is equal to the proportion of DNA thymine which is radioactive ([3H]thymine/total thymine). This equation may be rearranged to give ... 

Rates of DNA synthesis are sensitive to osmolality in both prokaryotic and eukaryotic cells. In bacteria, hyperosmolality led to a decrease in the frequency of initiation of DNA synthesis and, possibly for this reason, to an inhibition of cytokinesis (Meury, 1988). Glycine betaine was able to restore DNA synthesis, illustrating yet another benefit of relying on compatible organic osmolytes in osmoregulation. In mammalian cells, hyperosmolality has been found to both impede and increase DNA synthesis, depending on the cell types being studied and experimental conditions (Kiiltz, 2000). 

Specific inhibitions of DNA or protein biosyntheses have been illustrated in Figs. 3 and 4. Inhibition of protein synthesis results, after some delay, in the gradual decay of the rate of DNA synthesis as reviewed earlier in this section. The more complicated biosynthetic interrelations and consequences of inhibitions of RNA synthesis have also been reviewed with special reference to the action of actinomycin D34. In such instances it is important to concentrate on the early changes in rates of macromolecular syntheses after some delay, inhibitions of RNA synthesis will have produced shutdowns in protein and DNA syntheses. 

T cell activation results in the proliferation of primary T cells in culture, which can be measured directly by monitoring the rate of DNA synthesis. Metabolic incorporation of tritiated thymidine ( H-TdR) into cellular DNA is the most commonly used method, because it is highly sensitive and allows detection of poorly proliferating cells such as primary T cells. In addition, the H-TdR incorporation assay is time and cost effective and offers the possibility to screen the effects of several inhibitors, culture conditions, stimulations, etc. in a single 96-well plate. 

The rate of DNA synthesis can be measured by [ HJthymidine incorporation into the DNA of cells, or in a non-radioactive manner by bromodeoxyuridine (BrdU) incorporation using either a BrdU antibody in a colorimetric ELISA setting or by using FITC-conjugated BrdU and flow cytometry. 

Inhibition of DNA synthesis. The significance of the interaction of platinum compounds with cellular DNA is apparent from studies of drug effects on macromolecular synthesis. Cis-DDP selectively and persistently inhibits the rate of DNA synthesis as compared with effects on RNA and protein synthesis in cells in culture (60-63) and cells in vivo (64,65). 

In vitro tests of chick fibroblast cells showed that buffered ammonia-ammonium chloride solutions can induce clumping of chromosomes, inhibit spindle formation, and result in polyploidy (Rosenfeld 1932). Visek et al. (1972) noted reduced cell division in mouse fibroblasts cultured in media to which ammonia and ammonium chloride were added. The effect was noted in cultures irrespective of pH. Decreased rate of DNA synthesis was noted in mouse mucosal cells in the ileum and colon when serum levels were significantly elevated over normal levels these elevated levels were induced by intraperitoneal injection of urease or infusion of ammonium chloride (Zimber and Visek 1972a). 

Mouse ileal and colonic mucosa cells Decreased rate of DNA synthesis NH4CI NT + Zimber and Visek 1972a... 

Lomustine is a nitrosourea. Its mechanism of action involves the inhibition of both DNA and RNA synthesis through DNA alkylation. Lomustine has been shown to affect a number of cellular processes including RNA, protein synthesis, and the processing of ribosomal and nucleoplasmic messenger RNA DNA base component structure and the rate of DNA synthesis and DNA polymerase activity. It is cell-cycle nonspecific. It is indicated in the treatment of brain tumors and Hodgkin s disease in adults and children. 

Diamino-l,2,4-thiadiazole possesses radioprotective activity3, a correlation has been established466 between this property and a depressive effect on thymidine uptake on myelocytes, and on the rate of DNA synthesis in bone marrow and intestinal epithilium. The radioprotective activity of 5-amino-3-methylthio-l,2,4-thiadiazole has also been reported.467... 

A more dramatic difference is seen with ornithine decarboxylase (EC 4.1.1.17). This enzyme catcJyses the rale-limiting step in polyamine synthesis, and its activity in many tissues and organisms correlates well with the rate of DNA synthesis and cell proliferation. Its turnover is one of the most rapid of all enzymes, generally having a half-life of less than 20 min. Bullfield et al. (1988) have found a 20-fold higher activity in the skeletal muscle from the broiler strain compared with that in a layer strain of domestic fowl at one week of age. TTiis increased activity is almost certainly achieved by an increase in fcs with little change in fca-... 

---