Diisopropylamine dichloroacetate, a novel pyruvate dehydrogenase kinase 4 inhibitor, as a potential therapeutic agent for metabolic disorders and multiorgan failure in severe influenza.

Severe influenza is characterized by cytokine storm and multiorgan failure with metabolic energy disorders and vascular hyperpermeability. In the regulation of energy homeostasis, the pyruvate dehydrogenase (PDH) complex plays an important role by catalyzing oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid synthesis, and thus its activity is linked to energy homeostasis. The present study tested the effects of diisopropylamine dichloroacetate (DADA), a new PDH kinase 4 (PDK4) inhibitor, in mice with severe influenza. Infection of mice with influenza A PR/8/34(H1N1) virus resulted in marked down-regulation of PDH activity and ATP level, with selective up-regulation of PDK4 in the skeletal muscles, heart, liver and lungs. Oral administration of DADA at 12-h intervals for 14 days starting immediately after infection significantly restored PDH activity and ATP level in various organs, and ameliorated disorders of glucose and lipid metabolism in the blood, together with marked improvement of survival and suppression of cytokine storm, trypsin up-regulation and viral replication. These results indicate that through PDK4 inhibition, DADA effectively suppresses the host metabolic disorder-cytokine cycle, which is closely linked to the influenza virus-cytokine-trypsin cycle, resulting in prevention of multiorgan failure in severe influenza.

Blood lactate/ATP ratio, as an alarm index and real-time biomarker in critical illness.

OBJECTIVE: The acute physiology, age and chronic health evaluation (APACHE) II score and other related scores have been used for evaluation of illness severity in the intensive care unit (ICU), but there is still a need for real-time and sensitive prognostic biomarkers. Recently, alarmins from damaged tissues have been reported as alarm-signaling molecules. Although ATP is a member of the alarmins and its depletion in tissues closely correlates with multiple-organ failure, blood ATP level has not been evaluated in critical illness. To identify real-time prognostic biomarker of critical illness, we measured blood ATP levels and the lactate/ATP ratio (ATP-lactate energy risk score, A-LES) in critically ill patients. METHODS AND RESULTS: Blood samples were collected from 42 consecutive critically ill ICU patients and 155 healthy subjects. The prognostic values of blood ATP levels and A-LES were compared with APACHE II score. The mean ATP level (SD) in healthy subjects was 0.62 (0.19) mM with no significant age or gender differences. The median ATP level in severely ill patients at ICU admission was significantly low at 0.31 mM (interquartile range 0.25 to 0.44) than the level in moderately ill patient at 0.56 mM (0.38 to 0.70) (P<0.01). Assessment with ATP was further corrected by lactate and expressed as A-LES. The median A-LES was 2.7 (2.1 to 3.3) in patients with satisfactory outcome at discharge but was significantly higher in non-survivors at 38.9 (21.0 to 67.9) (P<0.01). Receiver operating characteristic analysis indicated that measurement of blood ATP and A-LES at ICU admission are as useful as APACHE II score for prediction of mortality. CONCLUSION: Blood ATP levels and A-LES are sensitive prognostic biomarkers of mortality at ICU admission. In addition, A-LES provided further real-time evaluation score of illness severity during ICU stay particularly for critically ill patients with APACHE II scores of ≥20.0.

An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells.

Firefly bioluminescence is widely used in the measurement of adenosine 5'-triphosphate (ATP) levels in biological materials. For such assays in tissues and cells, ATP must be extracted away from protein in the initial step and extraction efficacy is the main determinant of the assay accuracy. Extraction reagents recommended in the commercially available ATP assay kits are chaotropic reagents, trichloroacetic acid (TCA), perchloric acid (PCA), and ethylene glycol (EG), which extract nucleotides through protein precipitation and/or nucleotidase inactivation. We found that these reagents are particularly useful for measuring ATP levels in materials with relatively low protein concentrations such as blood cells, cultured cells, and bacteria. However, these methods are not suitable for ATP extraction from tissues with high protein concentrations, because some ATP may be co-precipitated with the insolubilized protein during homogenization and extraction, and it could also be precipitated by neutralization in the acid extracts. Here we found that a phenol-based extraction method markedly increased the ATP and other nucleotides extracted from tissues. In addition, phenol extraction does not require neutralization before the luciferin-luciferase assay step. ATP levels analyzed by luciferase assay in various tissues extracted by Tris-EDTA-saturated phenol (phenol-TE) were over 17.8-fold higher than those extracted by TCA and over 550-fold higher than those in EG extracts. Here we report a simple, rapid, and reliable phenol-TE extraction procedure for ATP measurement in tissues and cells by luciferase assay.

Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D).

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

Thiamine prevents obesity and obesity-associated metabolic disorders in OLETF rats.

We previously found that thiamine mitigates metabolic disorders in spontaneously hypertensive rats, harboring defects in glucose and fatty acid metabolism. Mutation of thiamine transporter gene SLC19A2 is linked to type 2 diabetes mellitus. The current study extends our hypothesis that thiamine intervention may impact metabolic abnormalities in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, exhibiting obesity and metabolic disorders similar to human metabolic syndrome. Male OLETF rats (4 wk old) were given free access to water containing either 0.2% or 0% of thiamine for 21 and 51 wk. At the end of treatment, blood parameters and cardiac functions were analyzed. After sacrifice, organs weights, histological findings, and hepatic pyruvate dehydrogenase (PDH) activity in the liver were evaluated. Thiamine intervention averted obesity and prevented metabolic disorders in OLETF rats which accompanied mitigation of reduced lipid oxidation and increased hepatic PDH activity. Histological evaluation revealed that thiamine alleviated adipocyte hypertrophy, steatosis in the liver, heart, and skeletal muscle, sinusoidal fibrosis with formation of basement membranes (called pseudocapillarization) which accompanied significantly reduced expression of laminin ß1 and nidogen-1 mRNA, interstitial fibrosis in the heart and kidney, fatty degeneration in the pancreas, thickening of the basement membrane of the vasculature, and glomerulopathy and mononuclear cell infiltration in the kidney. Cardiac and renal functions were preserved in thiamine treatment. Thiamine has a potential to prevent obesity and metabolic disorders in OLETF rats.

Prevention of incipient diabetic cardiomyopathy by high-dose thiamine.

Diabetic cardiomyopathy can progress toward overt heart failure with increased mortality. The hexosamine biosynthesis pathway has been implicated in signaling for fibrosis by the kidney. Thiamine (vitamin B(1)) is an indispensable coenzyme and required at intracellular glucose metabolism. In this study, we assessed if decrease of flux through the hexosamine biosynthesis pathway induced by high-dose thiamine therapy counteracts diabetes-induced cardiac fibrosis. The diabetes model used was the streptozotocin-induced diabetic rat. Normal control and diabetic rats were studied for 2 weeks with and without thiamine, and followings were analyzed; plasma biochemicals (total cholesterol and triglycerides), morphological changes, mRNA abundance relevant to cardiac failure (brain natriuretic peptide) and fibrosis (transforming growth factor-beta1, thrombospondine, fibronectin, plasminogen activator-I and connective tissue growth factor) as well as and matrix metalloproteinase activity were investigated. Thiamine repletion prevented diabetes-induced cardiac fibrosis without changes in plasma glucose concentration. This was achieved by prevention of thiamine depletion, increased pro-fibrotic mRNA abundance and decreased metalloproteinase activity in the heart of diabetic rats. O-glycosylated protein was significantly higher in the left ventricular of diabetic rats compared to control rats, which was decreased by thiamine administration. Thiamine repletion prevented diabetes-induced cardiac fibrosis in experimental diabetes, probably by suppression of hexosamine biosynthesis pathway.

DNA mismatch repair: molecular mechanism, cancer, and ageing.

DNA mismatch repair (MMR) proteins are ubiquitous players in a diverse array of important cellular functions. In its role in post-replication repair, MMR safeguards the genome correcting base mispairs arising as a result of replication errors. Loss of MMR results in greatly increased rates of spontaneous mutation in organisms ranging from bacteria to humans. Mutations in MMR genes cause hereditary nonpolyposis colorectal cancer, and loss of MMR is associated with a significant fraction of sporadic cancers. Given its prominence in mutation avoidance and its ability to target a range of DNA lesions, MMR has been under investigation in studies of ageing mechanisms. This review summarizes what is known about the molecular details of the MMR pathway and the role of MMR proteins in cancer susceptibility and ageing.

BRCA1 activates a G2-M cell cycle checkpoint following 6-thioguanine-induced DNA mismatch damage.

Human DNA mismatch repair (MMR) is involved in the response to certain chemotherapy drugs, including 6-thioguanine (6-TG). Consistently, MMR-deficient human tumor cells show resistance to 6-TG damage as manifested by a reduced G(2)-M arrest and decreased apoptosis. In this study, we investigate the role of the BRCA1 protein in modulating a 6-TG-induced MMR damage response, using an isogenic human breast cancer cell line model, including a BRCA1 mutated cell line (HCC1937) and its transfectant with a wild-type BRCA1 cDNA. The MMR proteins MSH2, MSH6, MLH1, and PMS2 are similarly detected in both cell lines. BRCA1-mutant cells are more resistant to 6-TG than BRCA1-positive cells in a clonogenic survival assay and show reduced apoptosis. Additionally, the mutated BRCA1 results in an almost complete loss of a G(2)-M cell cycle checkpoint response induced by 6-TG. Transfection of single specific small interfering RNAs (siRNA) against MSH2, MLH1, ATR, and Chk1 in BRCA1-positive cells markedly reduces the BRCA1-dependent G(2)-M checkpoint response. Interestingly, ATR and Chk1 siRNA transfection in BRCA1-positive cells shows similar levels of 6-TG cytotoxicity as the control transfectant, whereas MSH2 and MLH1 siRNA transfectants show 6-TG resistance as expected. DNA MMR processing, as measured by the number of 6-TG-induced DNA strand breaks using an alkaline comet assay (+/-z-VAD-fmk cotreatment) and by levels of iododeoxyuridine-DNA incorporation, is independent of BRCA1, suggesting the involvement of BRCA1 in the G(2)-M checkpoint response to 6-TG but not in the subsequent excision processing of 6-TG mispairs by MMR.

The interaction between two radiosensitizers: 5-iododeoxyuridine and caffeine.

5-Iododeoxyuridine (IUdR) and caffeine are recognized as potential radiosensitizers with different mechanisms of interaction with ionizing radiation (IR). To assess the interaction of these two types of radiosensitizers, we compared treatment responses to these drugs alone and in combination with IR in two p53-proficient and p53-deficient pairs of human colon cancer cell lines (HCT116 versus HCT116 p53-/- and RKO versus RKO E6). Based on clonogenic survival, the three single agents (IR, IUdR, and caffeine) as well as IUdR or caffeine combined with IR are less or equally effective in p53-deficient human tumor cells compared with p53-proficient tumor cells. However, using both radiosensitizers, a significantly greater radiosensitization was found in p53-deficient human tumor cells. To better understand the interaction of these two radiosensitizers, additional studies on DNA repair and cell cycle regulation were done. We found that caffeine enhanced IUdR-DNA incorporation and IUdR-mediated radiosensitization by partially inhibiting repair (removal) of IUdR in DNA. The repair of IR-induced DNA double-strand breaks was also inhibited by caffeine. However, these effects of caffeine on IUdR-mediated radiosensitization were not found in p53-proficient cells. Cell cycle analyses also showed a greater abrogation of IR-induced S- and G2-phase arrests by caffeine in p53-deficient cells, particularly when combined with IUdR. Collectively, these data provide the mechanistic bases for combining these two radiosensitizers to enhance tumor cytotoxicity. This differential dual mode of radiosensitization by combining IUdR and caffeine-like drugs (e.g., UCN-01) in p53-deficient human tumors may lead to a greater therapeutic gain.

CK2 inhibits apoptosis and changes its cellular localization following ionizing radiation.

In this study, we show that CK2 (casein kinase II, CKII) participates in apoptotic responses following ionizing radiation (IR). Using HeLa human cervical carcinoma cells, we find that transfection of small interfering RNA against the CK2 alpha and/or alpha' catalytic subunits results in enhanced apoptosis following IR damage as measured by flow cytometry techniques, compared with a control small interfering RNA. Within 2 to 6 hours of IR, CK2 alpha partially localizes to perinuclear structures, whereas a marked nuclear localization of alpha' occurs. Treatment with a pan-caspase inhibitor or transfection of ARC (apoptosis repressor with caspase recruitment domain) suppresses the apoptotic response to IR in the CK2-reduced cells, indicating involvement of caspases. Additionally, we find that CK2 alpha and/or alpha' reduction affects cell cycle progression independent of IR damage in this human cell line. However, the G2-M checkpoint following IR is not affected in CK2 alpha- and/or alpha'-reduced cells. Thus, our data suggest that CK2 participates in inhibition of apoptosis and negatively regulates caspase activity following IR damage.

Casein kinase 2 regulates both apoptosis and the cell cycle following DNA damage induced by 6-thioguanine.

PURPOSE: The purine antimetabolite, 6-thioguanine (6-TG), is an effective drug in the management of acute leukemias. In this study, we analyze the mechanisms of apoptosis associated with 6-TG treatment and casein kinase 2 (CK2 or CKII) in human tumor cells. EXPERIMENTAL DESIGN: Small interfering RNA and chemical CK2 inhibitors were used to reduce CK2 activity. Control and CK2 activity-reduced cells were cultured with 6-TG and assessed by flow cytometry to measure apoptosis and cell cycle profiles. Additionally, confocal microscopy was used to assess localization of CK2 catalytic units following 6-TG treatment. RESULTS: Transfection of small interfering RNA against the CK2 alpha and/or alpha' catalytic subunits results in marked apoptosis of HeLa cells following treatment with 6-TG. Chemical inhibitors of CK2 also induce apoptosis following 6-TG treatment. Apoptosis induced by 6-TG is similarly observed in both mismatch repair-proficient and -deficient HCT116 and HeLa cells. Concomitant treatment with a pan-caspase inhibitor or transfection of apoptosis repressor with caspase recruitment domain markedly suppresses the apoptotic response to DNA damage by 6-TG in the CK2-reduced cells, indicating caspase regulation by CK2. CK2 alpha relocalizes to the endoplasmic reticulum after 6-TG treatment. Additionally, transfection of Cdc2 with a mutation at Ser(39) to Ala, which is the CK2 phosphorylation site, partially inhibits cell cycle progression in G(1) to G(2) phase following 6-TG treatment. CONCLUSION: CK2 is essential for apoptosis inhibition following DNA damage induced by 6-TG, controlling caspase activity.

Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR-Chk1 pathway.

DNA mismatch repair (MMR) deficiency in human cancers is associated with resistance to a spectrum of clinically active chemotherapy drugs, including 6-thioguanine (6-TG). We and others have shown that 6-TG-induced DNA mismatches result in a prolonged G2/M cell cycle arrest followed by apoptosis in MMR(+) human cancer cells, although the signaling pathways are not clearly understood. In this study, we found that prolonged (up to 4 days) treatment with 6-TG (3microM) resulted in a progressive phosphorylation of Chk1 and Chk2 in MMR(+) HeLa cells, correlating temporally with a drug-induced G2/M arrest. Transfection of HeLa cells with small interfering RNA (siRNA) against the ataxia telangiectasia-related (ATR) kinase or against the Chk1 kinase destroyed the G2/M checkpoint and enhanced the apoptosis following 6-TG treatment. On the other hand, the induction of a G2/M population by 6-TG was similar in ATM(-/-) and ATM(+) human fibroblasts, suggesting that the ATM-Chk2 pathway does not play a major role in this 6-TG response. Our results indicate that 6-TG DNA mismatches activate the ATR-Chk1 pathway in the MMR(+) cells, resulting in a G2/M checkpoint response

Both DNA topoisomerase II-binding protein 1 and BRCA1 regulate the G2-M cell cycle checkpoint.

Cell cycle checkpoints play a central role in genomic stability. The human DNA topoisomerase II-binding protein 1 (TopBP1) protein contains eight BRCA1 COOH terminus motifs and shares similarities with Cut5, a yeast checkpoint Rad protein. TopBP1 also shares many features with BRCA1. We report that, when expression of TopBP1 protein is inhibited in BRCA1 mutant cells, mimicking a TopBP1, BRCA1 double-negative condition, the G(2)-M checkpoint is strongly abrogated and apoptosis is increased after ionizing radiation. However, a BRCA1-negative or a TopBP1-negative background resulted in only partial abrogation of the G(2)-M checkpoint. The BRCA1 mutant and TopBP1-reduced condition specifically destroys regulation of the Chk1 kinase but not the Chk2 kinase, suggesting involvement in the ataxia telangiectasia-related pathway. These results indicate that both TopBP1 and BRCA1 specifically regulate the G(2)-M checkpoint, partially compensating each function.

A Functional interaction between the human papillomavirus 16 transcription/replication factor E2 and the DNA damage response protein TopBP1.

The human papillomavirus (HPV) transcription/replication factor E2 is essential for the life cycle of HPVs. E2 protein binds to DNA target sequences in the viral long control regions to regulate transcription of the viral genome. It also enhances viral DNA replication by interacting with the viral replication factor E1 and recruiting it to the origin of replication and may also play a more direct role in replication. The cellular proteins with which E2 interacts to carry out these functions are largely unknown. To identify these proteins a yeast two-hybrid screen was carried out with the transcription/replication domain of HPV16 E2. This screen identified several candidate interacting partners for E2 including TopBP1 (topoisomerase II beta-binding protein 1). TopBP1 has eight BRCA1 carboxyl-terminal domains that are found in proteins regulating the DNA damage response, transcription, and replication. Here we demonstrate that HPV16 E2 and TopBP1 interact in vitro and in vivo and that TopBP1 can enhance the ability of E2 to activate transcription and replication. This is the first time that TopBP1 has been shown to function as a transcriptional coactivator and that E2 interacts with TopBP1. Removal of the amino-terminal domain of TopBP1 abolishes coactivation of transcription and replication. This interaction may have functional consequences upon the viral life cycle.

A DNA damage-regulated BRCT-containing protein, TopBP1, is required for cell survival.

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.