Incorporation into DNA

Trifluridine, C2qH22F2N20, (5-trifluoromethyl-2 -deoxyuridine [70-00-8] F TdU, 14) was first prepared (30) in 1962. It is used for topical therapy of herpes vims-infected eyes. It is especially usefiil for treating infections that are resistant to IdU therapy. Like IdU, trifluridine is incorporated into DNA in place of thymidine in both infected and uninfected cells. But it is 10 times more potent than IdU against herpes keratitis in rabbits and 10 times more soluble in water. Trifluridine is also usefiil in treating human cytomegalovims (HCMV), but its toxicity to bone marrow may limit its clinical use. 

BVdU is incorporated into DNA, but since it is phosphorylated specifically in the viraHy infected cell, incorporation is mainly confined to such cells. Evidence suggests that BVdU also inhibits the biosynthesis of HSV-1 glycoproteins, which may contribute to the inhibition of the HSV-1 vims (32). 

FIAC also strongly inhibits HCMV and Epstein-Barr vims (EBV) in vitro the two vimses known not to induce a specific viral thymidine kinase for their repHcation. However, HCMV may stimulate cellular kinases that can anabolize FIAC to its 5 -triphosphate, which specifically inhibits the HCMV-encoded DNA polymerase. This selective activity suggests that FIAC should be evaluated against HCMV infections. FIAC-ttiphosphate incorporated into DNA has shown strong in vitro activity against the DNA polymerases of human hepatitis B vims (HBV) and of woodchuck hepatitis vims (WHV) (37). 

The mode of action of PMEA may be quite similar to the mechanism by which (3)-HPMPA accomplishes its selective inhibitory activity against herpes vimses. Eor PMEA to reach its active triphosphate form, it needs only two phosphorylation steps. The triphosphate derivative of PMEA has a much stronger affinity for HIV-1 reverse transcriptase than for cellular DNA polymerases (175). Whether it is actually incorporated into DNA and terminates the growing DNA chain is currentiy under investigation. 

Decitabine (5-aza-deoxycytosine) is an analog of the nucleoside 2 -deoxycytidine. It is believed to exert its antineoplastic effects after phosphorylation and direct incorporation into DNA and by inhibition of the enzyme DNA methyltransferase, causing hypomethylation of DNA and cellular differentiation or apoptosis. DNA hypomethylation is achieved at concentrations below those required to significantly inhibit DNA synthesis, which may promote restoration of function to genes associated with control of cellular differentiation and proliferation. Cytotoxicity in rapidly dividing cells may also result from covalent adducts between DNA methyltransferase and decitabine. 

Synthetic analogs of purine and pyrimidine bases and their derivatives serve as anticancer dmgs either by inhibiting an enzyme of nucleotide biosynthesis or by being incorporated into DNA or RNA. 

To ensure that the inhibition of EGF binding by palytoxin was not a consequence of cell toxicity, the effect of palytoxin on DNA synthesis in Swiss 3T3 cells was monitored. When cells were incubated in the presence of palytoxin, 10% fetal calf serum, and H-thymidine for 19.5 hr, no depression in the extent of H-thymidine incorporation into DNA was detected up to 3.7 pM palytoxin (Table I). Although 11 pM palytoxin was toxic when present for a prolonged period, under the conditions of the assays described above no toxicity was detected (Table I). When cells were incubated in the presence of palytoxin, 0.1% fetal calf serum, and H-thymidine, palytoxin did not stimulate significant incorporation of H-thymidine into DNA. Thus, although it can modulate the EGF receptor system under these conditions, palytoxin alone does not appear to be mitogenic for Swiss 3T3 cells. 

Confluent Swiss 3T3 cells were serum-starved by incubation for 48 hr in DME containing 0.1% PCS. Cells were then incubated with the indicated compound at 37 C in the presence of H-thymidine, 0.1 or 10% PCS for 19.5 hr, washed, and then assayed for H-thymidine incorporation into DNA as described in Methods. 

LU Y p, LOU Y R, xiE J G, YEN p, HUANG M T and coNNEY A H (1997) Inhibitory effect of black tea on the growth of established skin tumors in mice effects on tumor size, apoptosis, mitosis and hromodeoxyuridine incorporation into DNA , Carcinogenesis, 18 (11), 2163-9. 

Subsequently, it has been found that 44 can be incorporated into DNA oligomers intact using alternative reagents and work-up procedures. However, the oligomers produced contain a 1 1 mixture of 44 45... 

Redox participants are chosen to facilitate spectroscopic, biochemical and electrochemical probing of DNA CT. These include metallointercalators, organic intercalators, and modified bases that possess useful, well-described, and varied redox, photophysical and photochemical properties (Table 1). Our probes are readily incorporated into DNA assemblies where CT distances ranging from 3.4 to 200 A and driving forces spanning over two volts can be modulated with certainty. Most importantly, all redox probes which afford fast and/or efficient CT through DNA are well-coupled to the 7r-stack. 

Incorporation of a flavin electron donor and a thymine dimer acceptor into DNA double strands was achieved as depicted in Scheme 5 using a complex phosphoramidite/H-phosphonate/phosphoramidite DNA synthesis protocol. For the preparation of a flavin-base, which fits well into a DNA double strand structure, riboflavin was reacted with benzaldehyde-dimethylacetale to rigidify the ribityl-chain as a part of a 1,3-dioxane substructure [49]. The benzacetal-protected flavin was finally converted into the 5 -dimethoxytri-tyl-protected-3 -H-phosphonate ready for the incorporation into DNA using machine assisted DNA synthesis (Scheme 5a). For the cyclobutane pyrimidine dimer acceptor, a formacetal-linked thymine dimer phosphoramidite was prepared, which was found to be accessible in large quantities [50]. Both the flavin base and the formacetal-linked thymidine dimer, were finally incorporated into DNA strands like 7-12 (Scheme 5c). As depicted in... 

Scheme 5b, solutions (pH=7.5) of these DNA strands were subsequently irradiated under anaerobic conditions, after reduction of the flavin with sodium dithionite [51]. HPLC analysis of samples removed from the assay solution during such an irradiation experiment showed clean transformation of the Tf=T-containing DNA strands into the repaired TfT-containing DNA strands (Fig. 2). These experiments showed for the first time that a reduced flavin, if incorporated into DNA, can inject an extra electron into a DNA du-...

Barber et al. (2006) Primary benign epithelial cells (PECs) isolated at the time of surgery in = 6) PSA-secreting LNCaP used as l-20pM lycopene (Sigma) THF 48 h Proliferation measured as BrdU incorporation into DNA... 

Ling YH, Chan JY, Beattie KL et al. Consequences of 6-thioguanine incorporation into DNA on polymerase, ligase, and endonuclease reactions. Mol Pharmacol 1992 42 802-807. 

Of the purine nucleosides, dATP may be derivatized at its N-6 position using a long linker arm terminating in a detectable group without losing the ability to be enzymatically incorporated into DNA probes. By contrast, if modification is done at the C-8 position of purine bases, DNA polymerase cannot by used to add the labeled monomer to an existing strand. C-8 derivatives, however, can be added at the 3 terminal using terminal transferase enzyme. 

The chemical modification of nucleic acids at specific sites within individual nucleotides or within oligonucleotides allows various labels to be incorporated into DNA or RNA probes. This labeling process can produce conjugates having sensitive detection properties for the localization or quantification of oligo binding to a complementary strand using hybridization assays. 

To establish whether rifaximin, like the other members of the rifamycin family [36, 58], specifically inhibits bacterial RNA synthesis the effect of this antibiotic as well as that of rifampicin and chloramphenicol on RNA (via 3H-uridine incorporation), DNA (via 3H-thymidine incorporation) and protein (via 35S-methionine incorporation) synthesis was studied in growing cultures of Escherichia coli [59], While chloramphenicol reduced protein synthesis, both rifaximin and rifampicin inhibited RNA synthesis in a concentration-dependent fashion. In contrast, none of them affected 3H-thymidine incorporation into DNA. These data suggest that rifaximin, like rifampicin, inhibits RNA synthesis by binding the (3 subunit of the bacterial DNA-dependent RNA polymerase [60],... 

Liu B, Bazan GC (2005) Methods for strand-specific DNA detection with cationic conjugated polymers suitable for incorporation into DNA chips and microarrays. Proc Natl Acad Sci USA 102 589-593... 

Three years later, Lajtha, Oliver, and Ellis performed similar studies with human bone-marrow cultures exposed to 32P, or 14C-adenine. Control smears were treated with M HC1 at 60 °C for 6.5 min to remove 32P not incorporated into DNA. Grain counts were made over individual nuclei so that the rate of uptake into DNA could be estimated. The cycle time for the dividing cells in the culture was 40-48 h. DNA synthesis took 12-15 h in the second half of the cycle and was divided from mitosis by a 3-4 h non-synthesizing period (G2). 

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