A Phase I dose-escalation study of third-line regorafenib with trifluridine/tipiracil in metastatic colorectal cancer.

Aim: To determine a recommended Phase II dose of the oral fluoropyrimidine trifluridine/tipiracil (FTD/TPI) combined with the multi-kinase inhibitor regorafenib (REG) in refractory metastatic colorectal cancer patients. Materials & methods: A conventional 3 + 3 dose finding design was used. FTD/TPI was administered on days 1-5 and 8-12 of a 28-day cycle, REG on days 2-22. Two dose levels were used: FTD/TPI 25 mg/m2 b.i.d. + REG 120 mg/d, then escalated to FTD/TPI 35 mg/m2 b.i.d. + REG 120 mg/d. Results: In total, 12 patients were treated at two dose levels. Three dose-limiting toxicities were observed; all were grade 3 hypertension causally attributed to REG. Recommended Phase II dose is FTD/TPI 25 mg/m2 b.i.d. + REG 120 mg/d. Median progression-free survival was 3.81 months (95% CI: 1.51-5.29), median OS 11.1 months (95% CI: 2.3-18.2). Conclusion: The combination of REG and FTD/TPI is feasible and safe. Efficacy signals exceed that of the single agents at acceptable toxicity levels and are clinically meaningful.

Lay abstract Many patients with metastatic colorectal cancer need a sequence of different treatments over time. Regorafenib and trifluridine/tipiracil (also called TAS-102) are two drugs which are both used late in this sequence of treatments, but there is no rule as to which should be used first. Both drugs have very different mechanisms of action, and it might be beneficial to patients to administer them both at the same time as a combination treatment, instead of sequential treatment. We therefore conducted a Phase Ib study with a small number of patients to investigate whether this combined treatment would be feasible and safe. The study was designed to test the drug combination at different doses, and we found that treatment with trifluridine/tipiracil at 25 mg/m² twice daily combined with regorafenib at 120 mg daily had acceptable side effects and is likely to be safe for use in future clinical trials. Efficacy results suggest that combined treatment with both drugs may extend patient's life span. However, these observations are preliminary and need testing in further clinical trials. Clinical trial registration: EudraCT 2016-001968-11; NCT03305913 (ClinicalTrials.gov).

Targeting Thymidine Phosphorylase With Tipiracil Hydrochloride Attenuates Thrombosis Without Increasing Risk of Bleeding in Mice.

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.

Self-assembled nanostructures of phosphomolybdate, nucleobase and metal ions synthesis and their in vitro cytotoxicity studies on cancer cell lines.

The ability of the multidentate nucleobases, adenine and thymine, to coordinate polyoxometalate and metal ions leading to the formation of self-assembled nanostructures and their strong cytotoxicity toward cancer cell lines have been demonstrated. A unique synthetic approach is developed to make a series of functional nanoscale hybrid materials consisting of nucleobases (adenine and thymine) and phosphomolybdic acid (PMA) through solid state chemical reaction and self-assembly process. Adenine was protonated through its ring nitrogen, while the ketone group in thymine was protonated during the addition of PMA to these nucleobases. The self-assembled nanostructures formed as a result of the electrostatic interaction between the protonated nucleobases and polyanionic PMA. To promote the base pairing between the nucleobases, chloroaurate ions and silver ions were added to each PMA/adenine and PMA/thymine nanostructures. The complexation between the nucleobases and the added metal ions was found to drive the formation of subsequent self-assembled nanostructures. All the materials were screened for their anticancer activity against breast (MDAMB-231) and prostate (PC-3) cancer cells, and non-cancerous keratinocyte cells HaCaT. PMA/adenine/[AuCl4]- and PMA/thymine/Ag+ nanostructures were found to have strong anti-cancer activity, while PMA/adenine/Ag+, PMA/thymine/[AuCl4]-, and PMA/pdenine, PMA/thymine nanostructures did not exhibit such activity. The unique redox properties of these materials and the self-assembly of the PMA and metal ions were the major factors responsible for the cytotoxicity. This unique approach of making functional nanomaterials incorporate the nucleobase, PMA and metal ions using solid state self-assembly and their anti-cancer applications are considered to be an effective approach for the development of inorganic nucleoside analogue bio-pharmaceutical agents.

Cationic nucleopeptides as novel non-covalent carriers for the delivery of peptide nucleic acid (PNA) and RNA oligomers.

Cationic nucleopeptides belong to a family of synthetic oligomers composed by amino acids and nucleobases. Their capability to recognize nucleic acid targets and to cross cellular membranes provided the basis for considering them as novel non-covalent delivery agents for nucleic acid pharmaceuticals. Herein, starting from a 12-mer nucleopeptide model, the number of cationic residues was modulated in order to obtain new nucleopeptides endowed with high solubility in acqueous medium, acceptable bio-stability, low cytotoxicity and good capability to bind nucleic acid. Two candidates were selected to further investigate their potential as nucleic acid carriers, showing higher efficiency to deliver PNA in comparison with RNA. Noteworthy, this study encourages the development of nucleopeptides as new carriers to extend the known strategies for those nucleic acid analogues, especially PNA, that still remain difficult to drive into the cells.

A new chitosan-thymine conjugate: synthesis, characterization and biological activity.

Conjugation of chitosan with nucleobases is expected to expand its not only antimicrobial activity but also anti-cancer activity. Here, we report the synthesis of a novel chitosan-thymine conjugate by the reaction between chitosan and thymine-1-yl-acetic acid followed by acylation. The synthesized conjugate was characterized by FTIR, XRD, (1)H NMR, TGA and SEM. The microbiological screening results demonstrated the antimicrobial activity of the conjugate against bacteria viz., Escherichia coli, Staphylococcus aureus, and fungi viz., Aspergillus niger. The chitosan-thymine conjugate also inhibited (p<0.05) the proliferation of human liver cancer cells (HepG2) in a dose-dependent manner but had no cellular toxicity in non-cancerous mouse embryonal fibroblast cells (NIH 3T3). Thus, the chitosan-nucleobase conjugate may open a new perspective in biomedical applications.

DNA polymerases beta and lambda bypass thymine glycol in gapped DNA structures.

Here we investigated the ability of the human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg(2+) or Mn(2+). Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases beta and lambda was examined. The results show that (i) DNA polymerase lambda is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase lambda to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase lambda, (iv) DNA polymerase beta catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase beta. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

Synthesis of 4,6-dideoxy-3-fluoro-2-keto-beta-D-glucopyranosyl analogues of 5-fluorouracil, N6-benzoyl adenine, uracil, thymine, N4-benzoyl cytosine and evaluation of their antitumor activities.

The synthesis of the unsaturated 4,6-dideoxy-3-fluoro-2-keto-beta-d-glucopyranosyl nucleosides of 5-fluorouracil (6a), N(6)-benzoyl adenine (6b), uracil (6c), thymine (6d) and N(4)-benzoyl cytosine (6e), is described. Monoiodination of compounds 1a,b, followed by acetylation, catalytic hydrogenation and finally regioselective 2'-O-deacylation afforded the partially acetylated dideoxynucleoside analogues of 5-fluorouracil (5a) and N(6)-benzoyl adenine (5b), respectively. Direct oxidation of the free hydroxyl group at the 2'-position of 5a,b, with simultaneous elimination reaction of the beta-acetoxyl group, afforded the desired unsaturated 4,6-dideoxy-3-fluoro-2-keto-beta-D-glucopyranosyl derivatives 6a,b. Compounds 1c-e were used as starting materials for the synthesis of the dideoxy unsaturated carbonyl nucleosides of uracil (6c), thymine (6d) and N(4)-benzoyl cytosine (6e). Similarly a protection-selective deprotection sequence followed by oxidation of the free hydroxyl group at the 2'-position of the dideoxy benzoylated analogues 9c-e with simultaneous elimination reaction of the beta-benzoyl group, gave the desired nucleosides 6c-e. None of the compounds was inhibitory to a broad spectrum of DNA and RNA viruses at subtoxic concentrations. The 5-fluorouracil derivative 6a was more cytostatic (50% inhibitory concentration ranging between 0.2 and 12 microM) than the other compounds.

Efficient formation of the tandem thymine glycol/8-oxo-7,8-dihydroguanine lesion in isolated DNA and the mutagenic and cytotoxic properties of the tandem lesions in Escherichia coli cells.

Reactive oxygen species can induce the formation of not only single-nucleobase lesions, which have been extensively studied, but also tandem lesions. Herein, we report a high frequency of formation of a type of tandem lesion, where two commonly observed oxidatively induced single-nucleobase lesions, that is, thymidine glycol (Tg) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), are vicinal to each other in calf thymus DNA upon exposure to Cu(II)/ascorbate along with H(2)O(2) or gamma-rays. We further explored how the tandem lesions perturb the efficiency and fidelity of DNA replication by assessing the replication products formed from the propagation, in Escherichia coli cells, of the single-stranded pYMV1 shuttle vectors containing two tandem lesions [5'-(8-oxodG)-Tg-3' and 5'-Tg-(8-oxodG)-3'] or an isolated Tg or 8-oxodG. The bypass efficiencies for the two tandem lesions were approximately one-half of those for the two isolated single-nucleobase lesions. The presence of an adjacent Tg could lead to significant increases in G-->T transversion at the 8-oxodG site as compared to that of a single 8-oxodG lesion; the frequencies of G-->T mutation were approximately 18, 32, and 28% for 8-oxodG that is isolated, in 5'-(8-oxodG)-Tg-3' and in 5'-Tg-(8-oxodG)-3', respectively. Moreover, both pol IV and pol V are involved, in part, in bypassing the Tg, either present alone or as part of the tandem lesions, in E. coli cells. Together, our results support that complex lesions could exert greater cytotoxic and mutagenic effects than when the composing individual lesions are present alone.

5'-O-Aliphatic and amino acid ester prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine (DOT): synthesis, anti-HIV activity, cytotoxicity and stability studies.

A series of (-)-beta-D-(2R,4R)-dioxolane-thymine-5'-O-aliphatic acid esters as well as amino acid esters were synthesized as prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine (DOT). The compounds were evaluated for anti-HIV activity against HIV-1(LAI) in human peripheral blood mononuclear (PBM) cells as well as for their cytotoxicity in PBM, CEM and Vero cells. Improved anti-HIV potency in vitro was observed for the compound 2-4 (5'-O-aliphatic acid esters) without increase in cytotoxicity in comparison to the parent drug. Chemical and enzymatic hydrolysis of the prodrugs was also studied, in which the prodrugs exhibited good chemical stability with the half-lives from 3 h to 54 h at pH 2.0 and 7.4 phosphate buffer. However, the prodrugs were relatively labile to porcine esterase with the half-lives from 12.3 to 48.0 min.

Exomethylene pyranonucleosides: efficient synthesis and biological evaluation of 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine.

A new series of unsaturated pyranonucleosides with an exocyclic methylene group and thymine as heterocyclic base have been designed and synthesized. d-Galactose (1) was readily transformed in three steps into the corresponding 1-(beta-d-galactopyranosyl)thymine (2). Selective protection of the primary hydroxyl group of 2 with a t-butyldimethylsilyl (TBDMS) group, followed by specific acetalation, and oxidation gave 1-(6-O-t-butyldimethylsilyl-3,4-O-isopropylidene-beta-d-lyxo-hexopyranosyl-2-ulose)thymine (5). Wittig reaction of the ketonucleoside 5, deprotection and tritylation of the 6'-hydroxyl group gave 1-(2-deoxy-2-methylene-6-O-trityl-beta-d-lyxo-hexopyranosyl)thymine (9). Exomethylene pyranonucleoside 9 was converted to the olefinic derivative 10, which after detritylation afforded the title compound 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine (11). These novel synthesized compounds were evaluated for antiviral activity against rotaviral infection and cytotoxicity in colon cancer. As compared to AZT, compounds 1-(2-deoxy-2-methylene-beta-d-lyxo-hexopyranosyl)thymine (7) and 1-(beta-d-lyxo-hexopyranosyl-2-ulose)thymine (8) showed to be more efficient, in rotavirus infections and in treatment of colon cancer.

Structure-cytotoxicity relationships for a series of HEPT derivatives.

Structure-cytotoxicity relationships were studied for a series of 90 HEPT derivatives by means of multiple linear regression (MLR) and artificial neural network (ANN) techniques. The values of log(1/CC50) (CC50=cytotoxic dose of compound required to reduce the proliferation of normal uninfected MT-4 cells by 50%) of the studied compounds were correlated with the descriptors encoding the chemical structures. Using the pertinent descriptors revealed by the regression analysis, a correlation coefficient of 0.935 ( s=0.149) for the training set ( n=81) was obtained for the ANN model with a 5-6-1 configuration. The results obtained from this study indicate that the cytotoxicity of HEPT derivatives is strongly dependent on hydrophobic factors, mainly log P(R1), and dependent on the steric factors, especially SigmaMW(R3+R4). Comparison of the descriptors' contribution obtained in MLR and ANN analysis shows that the contribution of some of the descriptors to cytotoxicity may be non-linear.

Mutagenesis by O(6)-methyl-, O(6)-ethyl-, and O(6)-benzylguanine and O(4)-methylthymine in human cells: effects of O(6)-alkylguanine-DNA alkyltransferase and mismatch repair.

Double-stranded and gapped shuttle vectors were used to study mutagenesis in human cells by O(6)-methyl (m(6)G)-, O(6)-ethyl (e(6)G)-, and O(6)-benzylguanine (b(6)G), and O(4)-methylthymine (m(4)T) when these bases were incorporated site-specifically in the ATG initiation codon of a lacZ' gene. Vectors were transfected into either human kidney cells (293) or colon tumor cells (SO) or into mismatch repair defective human colon tumor cells (H6 and LoVo). Cellular O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) was optionally inactivated by treating cells with O(6)-benzylguanine prior to transfection. In alkyltransferase competent cells, the mutagenicity of all the modified bases was substantially higher in gapped plasmids than in double-stranded plasmids. Alkyltransferase inactivation increased mutagenesis by the three O(6)-substituted guanines in both double-stranded and gapped plasmids but did not affect m(4)T mutagenesis. In the absence of alkyltransferase, mutagenesis by m(6)G and to a lesser extent e(6)G in double-stranded vectors was higher in the mismatch repair defective H6 and LoVo cells than in SO or 293 cells indicating that e(6)G as well as m(6)G were subject to mismatch repair processing in these cells. The level of mutagenesis by m(4)T and b(6)G was not affected by mismatch repair status. When incorporated in gapped plasmids and in the absence of alkyltransferase, the order of mutagenicity for the modified bases was m(4)T > e(6)G congruent with m(6)G > b(6)G. The O(6)-substituted guanines primarily produced G-->A transitions while m(4)T primarily produced T-->C transitions. However, m(4)T also produced a significant number of T-->A transversion mutations in addition to T-->C transitions in mismatch repair deficient LoVo cells.

Reactivity and mutagenicity of endogenous DNA oxopropenylating agents: base propenals, malondialdehyde, and N(epsilon)-oxopropenyllysine.

Malondialdehyde (MDA), a mutagenic product of lipid peroxidation, reacts with DNA to form the premutagenic lesion, pyrimido[1, 2-a]purin-10(3H)-one (M(1)G). M(1)G is present in normal human tissues, but the contribution of other endogenously produced MDA analogues is poorly understood. Oxidation of the DNA backbone can cause strand breaks and release base propenals, and MDA condensation with proteins yields N(epsilon)-oxopropenyllysine. Here we compare the M(1)G-forming ability and Salmonella typhimurium mutagenicity of MDA with adenine, thymine, and cytosine propenals and N(alpha)-acetyl-N(epsilon)-oxopropenyllysine methyl ester. Base propenals are 30-150 times more potent than MDA in M(1)G formation and are 30-60 times more mutagenic than MDA. In addition, the Fe-bleomycin complex, which generates base propenals, induced M(1)G, but gamma-radiation, which generates mostly MDA, did not. M(1)G formation by MDA and base propenals was concentration-dependent, time-dependent, and enhanced by acidic conditions. N(alpha)-Acetyl-N(epsilon)-oxopropenyllysine methyl ester was less reactive and less mutagenic than MDA. These differences in potency are consistent with differences in leaving group ability. This work supports a role for other MDA analogues, especially base propenals, in the formation of endogenous M(1)G adducts.

Comparison of mutagenesis by O6-methyl- and O6-ethylguanine and O4-methylthymine in Escherichia coli using double-stranded and gapped plasmids.

To compare mutagenesis by O6-methylguanine (m6G), O4-methylthymine (m4T) and O6-ethylguanine (e6G), and assess their genotoxicity in Escherichia coli, double-stranded and gapped plasmids were constructed containing a single m6G, e6G or m4T in the initiation codon (ATG) of a lacZ' gene. Modified base induced mutations were scored by the loss of lacZ' activity on X-gal-containing media resulting in formation of white or sectored (mutant) rather than blue (non-mutant) colonies. Genotoxicity experiments with gapped plasmids containing the modified bases indicated that m4T produced a greater number of bacterial colonies than m6G or e6G. m4T was more mutagenic (45% mutant colonies) than m6G (6%) or e6G (11%) in repair competent (w.t.) E. coli when incorporated in double-stranded plasmids. In gapped plasmids, m4T produced 99% mutant colonies (as was observed previously for e6G) in both w.t. E. coli or E. coli deficient in both O6-alkylguanine-DNA alkyltransferases as well as methylation-directed mismatch repair (ada(-)-ogt(-)-mutS[-]). m6G in gapped plasmids produced 62% mutant colonies in w.t. E. coli, but this percentage increased to 94% in the ada(-)-ogt(-)-mutS(-) strain. In double-stranded plasmids both m4T and m6G produced very similar distributions of mutant and non-mutant colonies in the ada(-)-ogt(-)-mutS(-) strain. These observations led to the conclusion that differences in the mutagenicity of m6G and m4T in w.t. E. coli were a result of preferential repair of m6G compared to m4T by alkyltransferase and mismatch repair mechanisms, and did not reflect differences in their respective coding efficiency or their inherent obstructiveness to DNA synthesis as was observed with e6G. The combination of alkyltransferase and mismatch repair was concluded to be primarily responsible for the apparent genotoxicity of m6G compared to m4T in double-stranded plasmids.

Relationship between calculus formation and carcinogenesis in the urinary bladder of rats administered the non-genotoxic agents thymine or melamine.

Urinary bladder lesions induced by administration of thymine or melamine were investigated in male F344 rats. Animals, 6 weeks old at the beginning of the experiment, received either 3.0 or 1.0% thymine or 3.0, 1.0 or 0.3% melamine in the diet for 36 weeks followed by a 4 week period without chemicals, the total observation time being 40 weeks. Carcinomas of the urinary bladder were observed in 1/20 (5%) rats in each of the 3.0% thymine and 1.0% melamine groups, and in 15/19 (79%) animals given the 3.0% melamine treatment. Papillomas were induced in 9/20 (45%), 12/19 (63%) and 1/20 (5%) among rats receiving the 3.0% thymine, 3.0% and 1.0% melamine treatments respectively. Exploratory laparotomy at the end of week 36 revealed calculus formation in 9/10 (90%), 10/10 (100%) and 7/10 (70%) rats in these groups. In the ureter of the 3.0% melamine treated group, a carcinoma and papillomas were induced in 1/19 (5%) and 3/19 (16%) animals respectively. However, no tumors were observed in the renal pelvis in any of the other treated groups. Thus, administration of 3.0% thymine in the diet results in calculus formation in the urinary bladder of F344 rats, and is associated with development of tumors. It was also confirmed that a 3.0% dose level of melamine in the diet induces tumors in both the urinary bladder and the ureter.

Use of shuttle vectors to study the molecular processing of defined carcinogen-induced DNA damage: mutagenicity of single O4-ethylthymine adducts in HeLa cells.

We developed a simian virus 40 based shuttle vector system to study the molecular consequences of distinct carcinogen-induced DNA lesions in human cells. To establish the mutagenicity of O4-ethylthymine adducts, oligonucleotides carrying a single O4-ethylthymine adduct at a unique position were ligated into the vector molecules. Following replication in HeLa cells on average 23% of the progeny molecules carried a mutation in the region of modification. The vast majority of these mutations represented single T----C transitions at the position of the modified base, most probably as a consequence of mispairing of the O4-ethylthymine residues during replication. To a minor extent the O4-ethylthymine adduct may also induce T----A transversions or double point mutations. The in vivo mutation frequency of the adduct was found to be comparable to that of a C-A mismatch at the same position, but was lower than that expected from in vitro experiments with adducted DNA templates and purified DNA polymerases.

Processing of ring saturation and fragmentation products of DNA thymine in Escherichia coli.

The biological processing of thymine ring saturation and fragmentation products is summarized in Table 1. The ring saturation product, thymine glycol, is a block to in vitro DNA synthesis, whereas the ring saturation product, dihydrothymine, is not. Both these lesions are recognized in vitro by endonucleases III and VIII. Since thymine glycol is a replicative block, it is a lethal lesion in vivo. The excision repair process for removal of thymine glycols from DNA is initiated in vivo by endonuclease III and is followed by the action of either exonuclease III or endonuclease IV. Thymine glycol is very efficiently bypassed by translesion bypass in both single and double stranded DNA, however, because thymine glycol templates an adenine (A) and retains pairing characteristics, it is at best a weakly mutagenic lesion. The thymine ring fragmentation product, urea, and apurinic/apyrimidinic (AP) sites are both strong blocks to in vitro DNA synthesis. Both are substrates in vitro for endonucleases III, IV, VIII and IX as well as exonuclease III. Both are lethal lesions in single stranded and double stranded phage transfecting DNA. The excision repair of urea residues and AP sites is initiated in vivo by either exonuclease III or endonuclease IV. Neither of these noninstructive lesions are efficiently bypassed by UV-induced translesion bypass, however, when bypass occurs mutations result. beta-ureidoisobutylic acid is also a block to DNA synthesis in vitro. DNA containing this lesion is a substrate for endonucleases VIII and IX. The biological processing of this ring open thymine fragmentation product has yet to be determined. Thus, these ring saturation and fragmentation products of thymine have provided a point of departure for understanding the biological processing of modified bases with altered pairing and/or stacking properties.

Mutagenic specificity of alkylated and oxidized DNA bases as determined by site-specific mutagenesis.

This work demonstrates the use of the tools of site-specific mutagenesis to study the mutagenic activity of two DNA adducts, O6-methylguanine and cis-thymine glycol. The former adduct is one of the methylated bases formed by carcinogenic and mutagenic alkylating agents. It was built into the single-stranded genome of bacteriophage M13 and replicated in Escherichia coli (E. coli). The mutation frequency of O6-methylguanine was 0.4% in physiologically normal cells. In cells in which the repair systems for O6-methylguanine were compromised by challenge with an alkylating agent, the mutation frequency rose to approximately 20%. DNA sequencing revealed that O6-methylguanine induced exclusively G----A transitions, which was most consistent with it pairing with thymine during DNA synthesis. The mutagenic effects also were investigated of cis-thymine glycol isomers, which are major, stable products of ionizing radiation and oxidative damage to DNA. By techniques similar to those employed for the study of O6-methylguanine mutagenesis, a single thymine glycol was situated in an M13 phage genome. The genome was replicated in E. coli that were physiologically normal, induced for SOS functions, or deficient in the nth gene product and, in all cases, the mutagenic processing of thymine glycol in vivo yielded mutant progeny phage at a frequency of 0.3-0.4%. All mutations occurred at the site that originally contained thymine glycol, and all were demonstrated by DNA sequencing to have resulted from targeted T----C transitions. These data suggest that thymine glycol pairs with guanine during replication.

Use of a dodecadeoxynucleotide to study repair of the O4-methylthymine lesion.

A dodecadeoxynucleotide of defined sequence containing O4-methylthymine was labeled at the 5' end with [32P] by the reaction with (gamma-32P]ATP and polynucleotide kinase. Extracts prepared from bacterial and mammalian sources such as the human cell lines, HeLa and HT29, and rat liver were incubated with the labeled, methylated dodecamer to determine the extent of repair of the lesion. The labeled, demethylated dodecamer was separated from the labeled methylated dodecamer on a reverse-phase column using a shallow methanol gradient. There was complete repair of O4-methylthymine by the E. coli alkyltransferase upon incubation for 4 h at 37 degrees C. There was no detectable amount of demethylated product formed upon incubation with HeLa or HT29 cell extract for the same incubation period. There was also no repair of the O4-methylthymine lesion in the presence of crude rat-liver extract. However, the rat-liver extract alone degraded the methylated substrate completely, and the assay had to be conducted in the presence of NaF, AMP and unlabeled, nonmethylated dodecamer to prevent this. The results obtained from this assay, which is at least an order of magnitude more sensitive than previous methods, are in agreement with previous results that the mammalian alkyltransferase is specific for O6-alkylguanine repair.