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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.