Base-Modified Nucleic Acids as a Powerful Tool for Synthetic Biology and Biotechnology.

The ability of various nucleoside triphosphate analogues of deoxyguanosine and deoxycytidine with 7-deazadeoxyadenosine (A1 ) and 5-chlorodeoxyuridine (T1 ) to serve as substrates for Taq DNA polymerase was evaluated. The triphosphate set composed of A1 , T1 , and 7-deazadeoxyguanosine with either 5-methyldeoxycytidine or 5-fluorodeoxycytidine was successfully employed in the polymerase chain reaction (PCR) of 1.5 kb fragments as well as random oligonucleotide libraries. Another effective combination of triphosphates for the synthesis of a 1 kb PCR product was A1 , T1 , deoxyinosine, and 5-bromodeoxycytidine. In vivo experiments using an antibiotic-resistant gene containing the latter set demonstrated that the bacterial machinery accepts fully modified sequences as genetic templates. Moreover, the ability of the base-modified segments to selectively protect DNA from cleavage by restriction endonucleases was shown. This approach can be used to regulate the endonuclease cleavage pattern.

Facile Access to Bromonucleosides Using Sodium Monobromoisocyanurate (SMBI).

Bromonucleosides constitute a significant class of molecules and are well known for their biological activity. 5-Bromouridine, 5-bromo-2'-deoxyuridine, 5-bromouridine-5'-triphosphate, and nucleotides containing 5-bromouridine have been tested and used for numerous biological studies. 8-Bromopurine nucleosides have been used as essential precursors for the synthesis of nucleosides with fluorescent properties. This unit describes protocols for the synthesis of bromonucleosides using sodium monobromoisocyanurate (SMBI) in a straightforward way. Reactions are carried out at room temperature, and aqueous solvent mixtures are used to dissolve the nucleosides. Sodium azide is used as catalyst for the bromination of pyrimidine nucleosides, and no catalyst is necessary for the bromination of purine nucleosides. Unprotected 2'-deoxy pyrimidine and 2'-deoxy purine nucleosides are treated with SMBI to afford C-5 bromo pyrimidine and C-8 bromo purine nucleosides, respectively. This methodology has been found to be efficient for the synthesis of bromonucleosides on gram scale with consistently high yields. © 2017 by John Wiley & Sons, Inc.

5-Bromo-2'-deoxycytidine-a potential DNA photosensitizer.

A double-stranded oligonucleotide, 80 base pairs in length, was multiply labeled with 5-bromo-2'-deoxycytidine (BrdC) using polymerase chain reaction (PCR). The modified oligonucleotide was irradiated with 300 nm photons and its damage was assayed by employing DHPLC, LC-MS and denaturing polyacrylamide gel electrophoresis (PAGE). Two types of damage were demonstrated, namely, single strand breaks (SSBs) and intrastrand cross-links (ICLs); the ICLs were in the form of d(G^C) and d(C^C) dimers. The former species are probably formed due to photoinduced electron transfer between the photoexcited BrdC and the ground state 2'-deoxyguanosine (dG), whereas the latter is a result of a cycloaddition reaction. Since SSBs and ICLs are potentially lethal to the cell, BrdC could be considered as a nucleoside with possible clinical applications.

The radiosensitivity of 5- and 6-bromocytidine derivatives--electron induced DNA degradation.

Halogenated nucleotides belong to the group of radiosensitizers that sensitize solid tumors when incorporated into genomic DNA. Here, we consider the propensity of two isomeric bromocytidine derivatives, 3',5'-diphosphates of 5-bromo-2'-deoxycytidine (5BrdCDP) and 6-bromo-2'-deoxycytidine (6BrdCDP), to be damaged by electrons - one of the most abundant products formed during radiotherapy. An intranucleotide degradation mechanism leading to phosphodiester bond breakage (a model of single strand breakage in labeled DNA) and a ketone derivative formation was found for 6BrdCDP, while for 5BrdCDP a similar mechanism is sterically hindered. 5BrdCDP is, therefore, suggested to undergo electron induced degradation involving hydrogen transfer from a neighboring nucleotide or environment.

HDAC inhibitors dysregulate neural stem cell activity in the postnatal mouse brain.

The mammalian central nervous system (CNS) undergoes significant expansion postnatally, producing astrocytes, oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy, a condition commonly treated with valproate/valproic acid (VPA), a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs, however, may be essential for the differentiation of distinct subpopulations of neurons and glia. Here, we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7-P14, combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG, rostral migratory stream (RMS), and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed, however, that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA, OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity, we found that short term treatment with VPA in vivo reduced neurosphere numbers and size, a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor, Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively, these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered, especially in individuals whose brains are actively undergoing key postnatal time windows of development.

Evaluation of (131/123)I-5-iodo-2'-deoxycytidine as a novel proliferation probe in a tumor mouse model.

This study evaluated a radioiodinated deoxycytidine analog, (131)I-5-iodo-2'-deoxycytidine ([(131)I]ICdR), as a novel proliferation probe and compared it with (131)I-5-iodo-2'-deoxyuridine ([(131)I]IUdR) in a NG4TL4 sarcoma-bearing mouse model. As an imaging agent, the biological characteristics of [(123)I]IUdR is not satisfactory due to its metabolic instability and short biological half-life in vivo. With [(123)I]ICdR/SPECT it was possible to clearly delineate the tumor lesion at 1h post-injection (tumor-to-muscle ratio 7.74) in tumor-bearing mice. The results of biodistribution were consistent with those observed in scintigraphic imaging. This study demonstrated that [(131)I]ICdR is a more promising SPECT probe than [(131)I]IUdR for imaging proliferation.

Monitoring tumor response with radiolabeled nucleoside analogs in a hepatoma-bearing mouse model early after doxisome(®) treatment.

PURPOSE: This study aims to demonstrate that 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) positron emission tomography (PET) is a promising modality for noninvasively monitoring the therapeutic efficacy of Doxisome(®) in a subcutaneous hepatoma mouse model. PROCEDURES: Male BALB/c nu/nu mice were inoculated with HepG2 hepatoma xenograft in the right flank. Doxisome(®) (5 mg/kg, three times a week for 2 weeks) was intravenously administrated for treatment. (18)F-FLT-microPET, biodistribution studies, and immunohistochemistry of Ki-67 were performed. RESULTS: A significant difference (p < 0.05) in tumor volume was observed on day 5 between treated and control groups. The tumor-to-muscle ratio derived from (18)F-FLT-PET and (123)I-ICdR-microSPECT images of Doxisome(®)-treated mice dropped from 12.55 ± 0.76 to 3.81 ± 0.31 and from 2.48 ± 0.42 to 1.59 ± 0.08 after a three-dose treatment, respectively, while that of the control group remained steady. The retarded proliferation rate of treated xenograft was confirmed by Ki-67 immunohistochemistry staining. CONCLUSIONS: This study clearly demonstrated that Doxisome(®) is an effective anti-cancer drug against the growth of HepG2 hepatoma and that (18)F-FLT-PET could provide early information of tumor response during treatment.

Synthesis, molecular conformation and activity against herpes simplex virus of (E)-5-(2-bromovinyl)-2'-deoxycytidine analogs.

Analogs of (E)-5-(2-bromovinyl)-2'-deoxycytidine (BrVdCyd) (1) by substitution at N(4) were synthesized to impart resistance against deamination. The anti-HSV-1 activity and solution conformation of these analogs were determined. N(4)-Acetyl-BrVdCyd (2) was a potent inhibitor of HSV-1 replication whereas N(4)-propanoyl-BrVdCyd (3) had good activity and N(4)-Butanoyl-BrVdCyd (4) had only low activity against HSV-1 replication. N(4)-Methyl-BrVdCyd (5) was devoid of activity against HSV-1.

Effects of the genotoxic agent 5-bromo-2'-deoxyuridine with or without pre-pubertal gonadectomy on brain monoamines and their metabolites in female rats.

A nucleotide analog 5-bromo-2'-deoxyuridine (BrdU) is a genotoxic compound. Previous studies have demonstrated that prenatal treatment of rodents with BrdU affects the development of cortical neurons, reduces dopamine levels, and elevates serotonin (5-HT) levels in the striatum in adult male offspring from BrdU-treated dams. Moreover, prenatal BrdU-treated rats show locomotor hyperactivity in both males and females. This study investigated sexual dimorphism in the effect of prenatal BrdU on monoamine metabolism. Sprague-Dawley rats were treated with BrdU on gestational days 9-15 (50mg/kg, i.p.) and monoamine metabolism was examined in female rats at 10 weeks of age. The influence of pre-pubertal gonadectomy on the effects of BrdU was also investigated. BrdU-treated females showed elevations of dopamine and 5-HT levels in the striatum; reductions in dopamine, dihydroxyphenylacetic acid, or homovanillic acid (HVA) in the hypothalamus or the midbrain; and elevated HVA and 5-HT in the hippocampus. Pre-pubertal gonadectomy had a suppressive effect on striatal dopamine levels in prenatal BrdU-treated females. The present data indicate sexual dimorphic effects of prenatal BrdU-treatment in striatal dopamine metabolism but not in serotonergic metabolism and suggest a contribution of the increasing gonadal hormones that accompany puberty to this sex difference.

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases.

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2'-deoxyguanosine (8-Br-dG), 8-bromo-2'-deoxyadenosine (8-Br-dA), and 5-bromo-2'-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.

Reaction of 2'-deoxycytidine with peroxynitrite in the presence of ammonium bromide.

Peroxynitrite, a reactive nitrogen species generated from nitric oxide and superoxide anion radical, is an endogenous potential risk factor for human cancer. When 2'-deoxycytidine was incubated with peroxynitrite at neutral pH and 37 degrees C, the reaction was greatly enhanced by the addition of ammonium bromide. Both ammonium ion and bromide ion were required to exert the enhancing effect. In addition to ammonium ion, methylamine and dimethylamine exerted the enhancing effect in the presence of bromide ion. Two major products were identified as 5-hydroxy-2'-deoxycytidine and 5-bromo-2'-deoxycytidine. Hypochlorite solution and bromine water reacted with 2'-deoxycytidine generating 5-hydroxy-2'-deoxycytidine and 5-bromo-2'-deoxycytidine in the presence of ammonium bromide with the yields similar to those of the reaction of peroxynitrite with ammonium bromide. Fenton reaction of 2'-deoxycytidine was suppressed by the addition of ammonium bromide. Nitrogen dioxide gas did not react with 2'-deoxycytidine in the presence or the absence of ammonium bromide. These results suggest that in the presence of ammonium ion or amines, bromide ion interacts with peroxynitrous acid, which is a protonated form of peroxynitrite, but not with hydroxyl radical or nitrogen dioxide generated by homolysis of peroxynitrous acid, to form hypobromous acid. In the presence of ammonium ion or amines, bromide ion may play a role in enhancing the genotoxic effects of peroxynitrite in humans.

Hypoxia-driven proliferation of embryonic neural stem/progenitor cells--role of hypoxia-inducible transcription factor-1alpha.

We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus in vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs in vitro and that hypoxia-inducible factor (HIF)-1alpha, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O(2), 10% O(2), and 3% O(2)) for 3 days. The results showed that hypoxia, especially 10% O(2), promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF-1alpha mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O(2). To further elucidate the potential role of HIF-1alpha in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1alpha, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1alpha mRNA was used to knock down HIF-1alpha. We found that overexpression of HIF-1alpha caused the same proliferative effect on NPCs under 20% O(2) as under 10% O(2). In contrast, knockdown of HIF-1alpha inhibited NPC proliferation induced by 10% O(2). These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1alpha was critical in this process.

Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine.

The replacement of thymidine with 5-bromo-2'-deoxyuridine (BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a BrdU or its closely related 5-bromo-2'-deoxycytidine (BrdC). Our results showed that two types of crosslink products could be induced from d(BrCG), d(BrUG), d(GBrU), or d(ABrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5' flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5' neighboring nucleoside was a dG than while it was a dA, and BrdC induced the formation of crosslink products much more efficiently than BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.

MIA (melanoma inhibitory activity) promoter mediated tissue-specific suicide gene therapy of malignant melanoma.

Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.

A novel concept of glomeruloid body formation in experimental cerebral metastases.

Glomeruloid bodies (GBs), tumor-associated vascular structures with a superficial resemblance to renal glomeruli, are important histopathological features of glioblastoma multiforme, but have also been described in other types of tumors and in cerebral metastases. The purpose of this study was to elucidate the pathogenesis of these lesions in an appropriate murine model of experimental brain metastases. To do so, we injected cells from 5 different tumor lines into the internal carotid artery of mice and investigated the development, composition, and fate of GBs growing within tumor nodules. Immunohistochemical analyses and 3-dimensional reconstruction of the cerebral vasculature showed clearly that the proliferating and migrating tumor cells pull the capillaries (and the adjacent capillary branching points) into the tumor cell nest. Initially, this process lead to the appearance of simple coiled vascular structures, which later developed into chaotic and tortuous vascular aggregates with multiple narrowed afferent and efferent microvessels. Despite the absence of sprouting angiogenesis, the very low level of endothelial cell proliferation index and the ruptures of the stretched and narrowed capillary segments observed frequently between the metastatic tumor nodules, necrosis was scarce in these lesions, implying that the blood supply from the multiple afferent microvessels and from the preexistent vascular bed sufficed to provide the tumor cells with oxygen and nutrients.

Identification and quantification of mutagenic halogenated cytosines by gas chromatography, fast atom bombardment, and electrospray ionization tandem mass spectrometry.

Oxidative modification of nucleic acids has been implicated in carcinogenesis. One potential mechanism involves halogenation by the myeloperoxidase and eosinophil peroxidase systems of phagocytes. In the current studies, three mass spectrometric methods for the in vitro and in vivo analysis of halogenated cytosines and deoxycytidines were compared: gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) with a quadrupole instrument, fast atom bombardment or electrospray ionization (ESI) tandem MS with a four-sector magnetic instrument, and liquid chromatography ESI tandem MS (HPLC-ESI-MS/MS) with an ion-trap instrument. GC-EI-MS with selected ion monitoring of dimethyl-tert-butylsilyl derivatives of nucleobases was the most sensitive method. High-energy collisionally induced dissociation MS/MS analysis with a four-sector magnetic instrument yielded detailed structural information about halogenated nucleoside adducts but required relatively large amounts of material. The most sensitive analysis of intact halogenated deoxycytidine was achieved with extracted ion chromatograms using HPLC-ESI-MS/MS with an ion-trap instrument. Our results indicate that GC-EI-MS is the methodology of choice for ultrasensitive analysis of halogenated cytosines. HPLC-ESI-MS/MS provides greater structural detail for these compounds and may rival GC-EI-MS in sensitivity with more advanced liquid chromatography applications. The mass spectrometric methods we have developed should be useful for evaluating the role of phagocyte-derived oxidants in halogenating nucleobases, nucleosides, and DNA at sites of inflammation.

Relation of distal nephron changes to proximal tubular damage in uranyl acetate-induced acute renal failure in rats.

AIMS: To elucidate the pathophysiological roles of the changes of distal nephron in uranyl acetate (UA)-induced acute renal failure (ARF), we investigated the relation of changes of constituent molecules in distal nephron to proximal tubular damage and repair in UA-treated rats. METHODS: ARF was induced in rats by intravenous injection of UA, and all rats received bromodeoxyuridine (BrdU) intraperitoneally 1 h before sacrifice. RESULTS: Proximal tubular damage with necrosis appeared as early as day 2, mainly in the outer stripe of outer medulla and reached a peak level at day 5. Slight cellular damage was evident in the distal nephron as early as day 3, reaching a peak level around day 9. Immunoreactive BrdU- or vimentin-positive regenerating proximal tubules (PT) appeared at day 2 and regenerating PT relining was almost completed by day 7. Immunostaining for EGF, which was constitutively expressed in the thick ascending limb (TAL) and distal convoluted tubule (DCT), diminished significantly as early as day 2, when PT regeneration became evident, and remained below normal levels until day 21. In contrast, slight immunoreactivity for EGF was observed in regenerated PT accompanying brush-border formation mainly after day 9, suggesting newly expressed EGF might contribute to PT maturation. Lectin staining or immunostaining for representative constituent molecules of the thin descending limb, TAL, DCT and collecting duct demonstrated marked and transient reduction after day 5. CONCLUSIONS: EGF was not associated with regenerating PT, but may be involved in the maturation of PT. Transient reduction in expression of constituent molecules of the distal nephron following the reduction in EGF could reflect dedifferentiation or phenotypic simplification during regenerative repair of PT in UA-induced ARF in rats.

X-ray analyses of d(GCGAAAGC) and d(GXGAAAGCT), where X = 2'-deoxy-5-iodocytidine.

DNA fragments containing a sequence d(GCGAAAGC) are known to be highly thermostable. To investigate the structural basis for such a specific property, crystallographic studies of the DNA octamer and a nanomer d(GXGAAAGCT) (X = 2'-deoxy-5-iodocytidine) have been performed. The present higher resolution X-ray analyses have shown that both DNA oligomers are stabilized respectively to form a zipper-type duplex homodimer. PAGE of these oligomers, however, indicates that they are monomeric even when their crystals were dissolved at room temperature.

Radiosensitization of rat glioma with bromodeoxycytidine and adenovirus expressing herpes simplex virus-thymidine kinase delivered by slow, rate-controlled positive pressure infusion.

Infection of rat RT2 glioma cells in vitro with an adenovirus (ADV-TK) expressing herpes simplex virus (HSV) thymidine kinase (TK) and subsequent exposure to 5-bromo-2'-deoxycytidine (BrdC), which is specifically incorporated into ADV-TK-infected cell DNA as 5-bromo-2'-deoxyuridine (BrdU), results in significant radiosensitization (sensitizer enhancement ratio: 1.4-2.3) compared with Ad beta gal-infected cells. Cell killing correlated well with increased BrdU DNA incorporation and with apoptosis. Whereas radiation (4 Gy) alone was relatively ineffective in inducing apoptosis, treatment with HSV-TK/BrdC resulted in BrdC dose- (10-100 microM) and time-dependent (24-48 hours) increases, and the combination of the two treatments produced a synergistic response (1.5- to 2-fold). To investigate the effects of the ADV-TK/BrdC treatment in vivo, RT2 cells were grown as soft tissue tumors in Fischer 344 rats and conditions for virus infusion were optimized by altering the volume and rate of infusion using a rate-controlled positive pressure device. We found that relatively large volumes (100-150 microL) of virus delivered at rates of < or = 1 microL/minute were optimal and gave uniform and reproducible results. Using these optimal infusion conditions, we were able to achieve 40% adenovirus infection in the tumor. Infection of RT2 tumors with ADV-TK and continuous administration of BrdC from an osmotic pump resulted in significant (.001 < P < .009) tumor regression 6 days after radiation (30 Gy delivered as 2 x 5 Gy over 3 days) compared with controls. In situ staining of sectioned tumors with anti-BrdU antibody or by high-performance liquid chromatography analysis of extracted and hydrolyzed tumor DNA confirmed that we obtained efficient and specific incorporation of BrdU into tumor cells. These results suggest that adenovirus-mediated delivery of HSV-TK in combination with BrdC and radiation can potentially be an efficient combination modality for the treatment of gliomas.

The anchor site of telomerase from Euplotes aediculatus revealed by photo-cross-linking to single- and double-stranded DNA primers.

Telomerase is a ribonucleoprotein enzyme that adds telomeric sequence repeats to the ends of linear chromosomes. In vitro, telomerase has been observed to add repeats to a DNA oligonucleotide primer in a processive manner, leading to the postulation of a DNA anchor site separate from the catalytic site of the enzyme. We have substituted photoreactive 5-iododeoxypyrimidines into the DNA oligonucleotide primer d(T4G4T4G4T4G2) and, upon irradiation, obtained cross-links with the anchor site of telomerase from Euplotes aediculatus nuclear extract. No cross-linking occurred with a primer having the same 5' end and a nontelomeric 3' end. These cross-links were shown to be between the DNA primer and (i) a protein moiety of approximately 130 kDa and (ii) U51-U52 of the telomerase RNA. The cross-linked primer could be extended by telomerase in the presence of [alpha-32P]dGTP, thus indicating that the 3' end was bound in the enzyme active site. The locations of the cross-links within the single-stranded primers were 20 to 22 nucleotides upstream of the 3' end, providing a measure of the length of DNA required to span the telomerase active and anchor sites. When the single-stranded primers are aligned with the G-rich strand of a Euplotes telomere, the cross-linked nucleotides correspond to the duplex region. Consistent with this finding, a cross-link to telomerase was obtained by substitution of 5-iododeoxycytidine into the CA strand of the duplex region of telomere analogs. We conclude that the anchor site in the approximately 130-kDa protein can bind duplex as well as single-stranded DNA, which may be critical for its function at chromosome ends. Quantitation of the processivity with single-stranded DNA primers and double-stranded primers with 3' tails showed that only 60% of the primer remains bound after each repeat addition.