Determining Candidate Single Nucleotide Polymorphisms in Acquired Laryngotracheal Stenosis.

OBJECTIVES/HYPOTHESIS: Despite wide adoption of strategies to prevent injury from prolonged intubation and tracheotomy, acquired laryngotracheal stenosis (ALTS) has not disappeared. ALTS' persistence may be due to patient factors that confer unique susceptibility for some. We sought to identify genetic markers in genes associated with wound healing that could be associated with ALTS. STUDY DESIGN: Case-control study. METHODS: One hundred thirty-eight patients were recruited, 53 patients with ALTS and 85 control patients who underwent intubation or tracheotomy without evidence of ALTS. The patients' DNA was isolated from whole blood. Custom primers were designed, and the TaqMan assay employing allele-specific polymerase chain reaction was used to interrogate single nucleotide polymorphisms (SNPs) rs1799750, rs522616, rs2276109, rs2569190, rs1800469, and rs1024611 of candidate wound healing genes MMP1, MMP3, MMP12, CD14, TGFß1, and MCP1, respectively. A logistic regression model was used to examine the association of candidate gene polymorphisms with the presence or absence of ALTS. RESULTS: All 138 patients were successfully genotyped. No significant association was found between candidate SNPs and development of ALTS in the overall group. However, subgroup analysis within each ethnicity identified SNPs that are associated with ALTS depending upon the ethnic background. CONCLUSIONS: Patient factors such as variations in wound healing due to functional SNPs may shed light on the development of ALTS. There may be a difference in susceptibility to developing ALTS in different ethnic backgrounds. These preliminary findings need to be corroborated in larger population studies. LEVEL OF EVIDENCE: 3b. Laryngoscope, 128:E111-E116, 2018.

Disruption of NAD(+) binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions.

AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD(+) cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD(+) binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD(+) binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD(+) (Km = 741 ± 257 µmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD(+) binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD(+) binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD(+) binding center in GAPDH interactions with its intranuclear partners.

Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication.

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand-protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (Kd=4.4µM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.

Establishing a model for assessing DNA damage in murine brain cells as a molecular marker of chemotherapy-associated cognitive impairment.

AIMS: Chemotherapy-associated cognitive impairment often follows cancer chemotherapy. We explored chemotherapy-induced DNA damage in the brain cells of mice treated with 5-fluorouracil (5FU), an antineoplastic agent, to correlate the extent of DNA damage to behavioral functioning in an autoshaping-operant mouse model of chemotherapy-induced learning and memory deficits (Foley et al., 2008). MAIN METHODS: Male, Swiss-Webster mice were injected once with saline or 75 mg/kg 5FU at 0, 12, and 24h and weighed every 24h. Twenty-four h after the last injection, the mice were tested in a two-day acquisition and the retention of a novel response task for food reinforcement. Murine brain cells were analyzed for the presence of single- and double-strand DNA breaks by the single cell gel electrophoresis assay (the Comet assay). KEY FINDINGS: We detected significant differences (p<0.0001) for all DNA damage characteristics (DNA "comet" tail shape, migration pattern, tail moment and olive moments) between control mice cohort and 5FU-treated mice cohort: tail length - 119 vs. 153; tail moment - 101 vs. 136; olive moment - 60 vs. 82, correspondingly. We found a positive correlation between increased response rates (r=0.52, p<0.05) and increased rate of errors (r=0.51, p<0.05), and DNA damage on day 1. For all 15 mice (saline-treated and 5FU-treated mice), we found negative correlations between DNA damage and weight (r=-0.75, p<0.02). SIGNIFICANCE: Our results indicate that chemotherapy-induced DNA damage changes the physiological status of the brain cells and may provide insights to the mechanisms for cognitive impairment after cancer chemotherapy.

Cytotoxicity of chemotherapeutic agents in glyceraldehyde-3-phosphate dehydrogenase-depleted human lung carcinoma A549 cells with the accelerated senescence phenotype.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a central role in glycolysis. Because cancer cells rely on aerobic glycolysis rather than oxidative phosphorylation, GAPDH-depleting agents have a therapeutic potential to impede cancer cell proliferation. Knockdown of GAPDH by RNA interference induced the accelerated senescent phenotype in A549 cells, suggesting that GAPDH is a potential molecular target for combination chemotherapy. The cytotoxic effects of a panel of anticancer drugs, 5-fluorouracil, 5-fluorouridine, 5-fluorodeoxyuridine, 6-thioguanine, cytarabine, fludarabine, cladribine, clofarabine, 2-chloroadenosine, and doxorubicin, were assessed in GAPDH-depleted A549 cells using a cell proliferation assay. GAPDH-depleted A549 cells, when compared with control cells, exhibited increased chemoresistance to several antimetabolite agents including cytarabine [inhibitory concentration 50 (IC50) 1.7±0.3 vs. 0.03±0.02 µmol/l], 2-chloroadenosine (IC50 7.1±1.8 vs. 1.5±0.6 µmol/l), 6-thioguanine (IC50 7.5±1.6 vs. 1.4±0.5 µmol/l), 5-fluorouracil (IC50 13.2±2.5 vs. 3.0±0.7 µmol/l), and 5-fluorodeoxyuridine (IC50 >100 vs. 3.7±0.9 µmol/l), which we designated as group A agents. In contrast, GAPDH-deficient and GAPDH-proficient cells were equally sensitive to group B agents including doxorubicin (IC50 0.05±0.02 vs. 0.04±0.02 µmol/l), fludarabine (IC50 18.5±2.3 vs. 15.7±2.8 µmol/l), 5-fluorouridine (IC50 0.1±0.03 vs. 0.1±0.03 µmol/l), clofarabine (IC50 0.7±0.3 vs. 0.5±0.3 µmol/l), and cladribine (IC50 0.5±0.1 vs. 0.5±0.2 µmol/l). After treatment with group B agents at concentrations equivalent to 7-10-fold the IC50 value, the fraction of apoptotic cells in GAPDH-depleted, senescent A549 cells was similar to that in GAPDH-proficient cells. Our study identified the antimetabolite drugs active in senescent cells that can be used in combination with GAPDH inhibitors in cancer treatment. GAPDH-targeted combination therapy is a novel strategy to control the proliferation of tumor cells.

Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells.

DNA mismatch repair enzymes (for example, MSH2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. We found that leukemia cells from a substantial proportion of children (∼11%) with newly diagnosed acute lymphoblastic leukemia have low or undetectable MSH2 protein levels, despite abundant wild-type MSH2 mRNA. Leukemia cells with low levels of MSH2 contained partial or complete somatic deletions of one to four genes that regulate MSH2 degradation (FRAP1 (also known as MTOR), HERC1, PRKCZ and PIK3C2B); we also found these deletions in individuals with adult acute lymphoblastic leukemia (16%) and sporadic colorectal cancer (13.5%). Knockdown of these genes in human leukemia cells recapitulated the MSH2 protein deficiency by enhancing MSH2 degradation, leading to substantial reduction in DNA mismatch repair and increased resistance to thiopurines. These findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating MSH2 degradation result in undetectable levels of MSH2 protein in leukemia cells, DNA mismatch repair deficiency and drug resistance.

Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-ß-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of α subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

Glyceraldehyde 3-phosphate dehydrogenase depletion induces cell cycle arrest and resistance to antimetabolites in human carcinoma cell lines.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a multifunctional protein that acts at the intersection of energy metabolism and stress response in tumor cells. To elucidate the role of GAPDH in chemotherapy-induced stress, we analyzed its activity, protein level, intracellular distribution, and intranuclear mobility in human carcinoma cells A549 and UO31 after treatment with cytarabine, doxorubicin, and mercaptopurine. After treatment with cytosine arabinoside (araC), enzymatically inactive GAPDH accumulated in the nucleus. Experiments on fluorescence recovery after photobleaching with green fluorescent protein-GAPDH fusion protein in the live cells treated with araC demonstrated reduced mobility of green fluorescent protein-GAPDH inside the nucleus, indicative of interactions with nuclear macromolecular components after genotoxic stress. Depletion of GAPDH with RNA interference stopped cell proliferation, and induced cell cycle arrest in G(1) phase via p53 stabilization, and accumulation of p53-inducible CDK inhibitor p21. Neither p21 accumulation nor cell cycle arrest was detected in GAPDH-depleted p53-null NCI-H358 cells. GAPDH-depleted A549 cells were 50-fold more resistant to treatment with cytarabine (1.68 +/- 0.182 microM versus 0.03 +/- 0.015 microM in control). Depletion of GAPDH did not significantly alter cellular sensitivity to doxorubicin (0.05 +/- 0.023 microM versus 0.035 +/- 0.0154 microM in control). Induction of cell cycle arrest in p53-proficient carcinoma cells via GAPDH abrogation suggests that GAPDH-depleting agents may have a cytostatic effect in cancer cells. Our results define GAPDH as an important determinant of cellular sensitivity to antimetabolite chemotherapy because of its regulatory functions.

Chromatin-associated proteins HMGB1/2 and PDIA3 trigger cellular response to chemotherapy-induced DNA damage.

The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 micromol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no gamma-H2AX accumulated after 24-72 hours of incubation with 1 micromol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.

TPMT genetic variations in populations of the Russian Federation.

BACKGROUND: Polymorphisms that reduce the activity of thiopurine S-methyltransferase (TPMT) cause adverse reactions to conventional doses of thiopurines, routinely used for antileukemic and immunosuppressive treatment. There are more than 20 variant alleles of TPMT that cause decreased enzymatic activity. We studied the most common variant alleles of TPMT and their frequency distribution in a large cohort of multiracial residents in the Russian Federation and compared their frequencies in children with and without malignancy to determine whether TPMT gene abnormality is associated with hematologic malignancy. PROCEDURE: The TPMT biochip was used to detect 6 TPMT single nucleotide polymorphisms (SNPs) corresponding to 7 TPMT-deficiency alleles (TPMT*2, TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8). We analyzed allele frequencies in the whole cohort, the childhood cancer group, and the non-cancer group. We also characterized disease features and outcome according to the presence of TPMT SNPs in children with acute lymphoblastic leukemia (ALL). RESULTS: Fifty-five (5.5%) study participants overall had heterozygous TPMT genotypes (1 variant and 1 wild-type allele): TPMT*1/*3A (n = 45; 4.5%), TPMT*1/*3C (n = 8; 0.8%), and TPMT*1/*2 (n = 2; 0.2%). TPMT SNPs were more frequent in children with hematologic malignancy than in other participants (7.5% vs. 4.0%, P = 0.02). We found no significant association between TPMT SNPs and ALL treatment outcome (median follow-up, 31.3 months). CONCLUSIONS: TPMT*3A is the most prevalent variant allele in the Russian Federation. The estimated frequency of variant alleles in the study cohort (5.5%) was similar to that observed in the White populations in the U.S. and Eastern Europe.

High mobility group protein B1 is an activator of apoptotic response to antimetabolite drugs.

We explored the role of a chromatin-associated nuclear protein high mobility group protein B1 (HMGB1) in apoptotic response to widely used anticancer drugs. A murine fibroblast model system generated from Hmgb1(+)(/)(+) and Hmgb1(-/-) mice was used to assess the role of HMGB1 protein in cellular response to anticancer nucleoside analogs and precursors, which act without destroying the integrity of DNA. Chemosensitivity experiments with 5-fluorouracil, cytosine arabinoside (araC), and mercaptopurine (MP) demonstrated that Hmgb1(-/-) mouse embryonic fibroblasts (MEFs) were 3 to 10 times more resistant to these drugs compared with Hmgb1(+)(/)(+) MEFs. Hmgb1-deficient cells showed compromised cell cycle arrest and reduced caspase activation after treatment with MP and araC. Phosphorylation of p53 at Ser12 (corresponding to Ser9 in human p53) and Ser18 (corresponding to Ser15 in human p53), as well as phosphorylation of H2AX after drug treatment, was reduced in Hmgb1-deficient cells. trans-Activation experiments demonstrated diminished activation of proapoptotic promoters Bax, Puma, and Noxa in Hmgb1-deficient cells after treatment with MP or araC, consistent with reduced transcriptional activity of p53. We have demonstrated for the first time that Hmgb1 is an essential activator of cellular response to genotoxic stress caused by chemotherapeutic agents (thiopurines, cytarabine, and 5-fluorouracil), which acts at early steps of antimetabolite-induced stress by stimulating phosphorylation of two DNA damage markers, p53 and H2AX. This finding makes HMGB1 a potential target for modulating activity of chemotherapeutic antimetabolites. Identification of proteins sensitive to DNA lesions that occur without the loss of DNA integrity provides new insights into the determinants of drug sensitivity in cancer cells.

Rapid genotyping of common deficient thiopurine S-methyltransferase alleles using the DNA-microchip technique.

Thiopurine drugs are metabolized, in part, by S-methylation catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low or undetectable TPMT activity are at high risk of severe, potentially fatal hematopoietic toxicity when they are treated with standard doses of thiopurines. As human TPMT activity is controlled by a common genetic polymorphism, it is an excellent candidate for the clinical application of pharmacogenetics. Here, we report a new molecular approach developed to detect point mutations in the TPMT gene that cause the loss of TPMT activity. A fluorescently labeled amplified DNA is hybridized with oligonucleotide DNA probes immobilized in gel pads on a biochip. The specially designed TPMT biochip can recognize six point mutations in the TPMT gene and seven corresponding alleles associated with TPMT deficiency: TPMT*2; TPMT*3A, TPMT*3B, TPMT*3C, TPMT*3D, TPMT*7, and TPMT*8. The effectiveness of the protocol was tested by genotyping 58 samples of known genotype. The results showed 100% concordance between the biochip-based approach and the established PCR protocol. The genotyping procedure is fast, reliable and can be used for rapid screening of inactivating mutations in the TPMT gene. The study also provides the first data on the frequency of common TPMT variant alleles in the Russian population, based on a biochip analysis of 700 samples. TPMT gene mutations were identified in 44 subjects; genotype *1/*3A was most frequent.

A novel CRM1-mediated nuclear export signal governs nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase following genotoxic stress.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.

Msh2 deficiency attenuates but does not abolish thiopurine hematopoietic toxicity in msh2-/- mice.

The amount of MSH2 protein, a major component of the mismatch repair system, was found to differ >10-fold in leukemia cells from children with newly diagnosed acute lymphoblastic leukemia, with a subgroup of patients (17%) having undetectable MSH2 protein. We therefore used a murine Msh2 knockout model to elucidate the in vivo importance of MSH2 protein expression in determining thiopurine hematopoietic cytotoxicity. After mercaptopurine (MP) treatment (30 mg/kg/day for 14 days), there was a significantly greater decrease in circulating leukocytes in Msh2+/+ and Msh2+/- mice when compared with Msh2-/- mice (p < 0.002). Likewise, the decrease in erythrocyte counts was more prominent in mice with at least one functional Msh2 allele. MP doses of more than 50 mg/kg/day for 14 days resulted in treatment-related deaths, but Msh2-/- mice had a significant survival advantage (p = 0.02). Murine embryonic fibroblasts (MEFs) from Msh2+/+ mice also exhibited increased sensitivity to MP when compared with MEFs from Msh2-/- mice (IC50, 3.8 +/- 0.1 microM versus 11.9 +/- 1.3 microM, p < 0.001). After MP treatment, deoxythioguanosine incorporation into DNA was similar in mice and MEFs with each of the Msh2 genotypes. Electromobility shift assay experiments identified an Msh2-containing GT- or GST-DNA-nuclear protein complex in Msh2+/+ but not Msh2-/- MEFs. Together, these findings establish that hematopoietic toxicity in vivo after treatment with mercaptopurine is attenuated but not abolished by MSH2 deficiency.

A nuclear protein complex containing high mobility group proteins B1 and B2, heat shock cognate protein 70, ERp60, and glyceraldehyde-3-phosphate dehydrogenase is involved in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues.

Thiopurine treatment of human leukemia cells deficient in components of the mismatch repair system (Nalm6) initiated apoptosis after incorporation into DNA, as revealed by caspase activation and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. To elucidate the cellular sensor(s) responsible for recognition of DNA damage in cells with an inactive mismatch repair system, we isolated a multiprotein nuclear complex that preferentially binds DNA with thioguanine incorporated. The components of this nuclear multiprotein complex, as identified by protein mass spectroscopy, included high mobility group proteins 1 and 2 (HMGB1, HMGB2), heat shock protein HSC70, protein disulfide isomerase ERp60, and glyceraldehyde 3-phosphate dehydrogenase. The same complex was also shown to bind synthetic oligodeoxyribonucleotide duplexes containing the nonnatural nucleosides 1-beta-D-arabinofuranosylcytosine or 5-fluoro-2'-deoxyuridine. Fibroblast cell line derived from Hmgb1(-/-) murine embryos had decreased sensitivity to thiopurines, with an IC(50) 10-fold greater than Hmgb1-proficient cells (P < 0.0001) and exhibited comparable sensitivity to vincristine, a cytotoxic drug that is not incorporated into DNA. These findings indicate that the HMGB1-HMGB2-HSC70-ERp60-glyceraldehyde 3-phosphate dehydrogenase complex detects changes in DNA structure caused by incorporation of nonnatural nucleosides and is a determinant of cell sensitivity to such DNA modifying chemotherapy.

Structure and dynamics of thioguanine-modified duplex DNA.

Mercaptopurine and thioguanine, two of the most widely used antileukemic agents, exert their cytotoxic, therapeutic effects by being incorporated into DNA as deoxy-6-thioguanosine. However, the molecular mechanism(s) by which incorporation of these thiopurines into DNA translates into cytotoxicity is unknown. The solution structure of thioguanine-modified duplex DNA presented here shows that the effects of the modification on DNA structure were subtle and localized to the modified base pair. Specifically, thioguanine existed in the keto form, formed weakened Watson-Crick hydrogen bonds with cytosine and caused a modest approximately 10 degrees opening of the modified base pair toward the major groove. In contrast, thioguanine significantly altered base pair dynamics, causing an approximately 80-fold decrease in the base pair lifetime with cytosine compared with normal guanine. This perturbation was consistent with the approximately 6 degrees C decrease in DNA melting temperature of the modified oligonucleotide, the 1.13 ppm upfield shift of the thioguanine imino proton resonance, and the large increase in the exchange rate of the thioguanine imino proton with water. Our studies provide new mechanistic insight into the effects of thioguanine incorporation into DNA at the level of DNA structure and dynamics, provide explanations for the effects of thioguanine incorporation on the activity of DNA-processing enzymes, and provide a molecular basis for the specific recognition of thioguanine-substituted sites by proteins. These combined effects likely cooperate to produce the cellular responses that underlie the therapeutic effects of thiopurines.