The cGAS-STING signaling pathway is modulated by urolithin A.

During aging, general cellular processes, including autophagic clearance and immunological responses become compromised; therefore, identifying compounds that target these cellular processes is an important approach to improve our health span. The innate immune cGAS-STING pathway has emerged as an important signaling system in the organismal defense against viral and bacterial infections, inflammatory responses to cellular damage, regulation of autophagy, and tumor immunosurveillance. These key functions of the cGAS-STING pathway make it an attractive target for pharmacological intervention in disease treatments and in controlling inflammation and immunity. Here, we show that urolithin A (UA), an ellagic acid metabolite, exerts a profound effect on the expression of STING and enhances cGAS-STING activation and cytosolic DNA clearance in human cell lines. Animal laboratory models and limited human trials have reported no obvious adverse effects of UA administration. Thus, the use of UA alone or in combination with other pharmacological compounds may present a potential therapeutic approach in the treatment of human diseases that involves aberrant activation of the cGAS-STING pathway or accumulation of cytosolic DNA and this warrants further investigation in relevant transgenic animal models.

Influence of Nodal signalling on pluripotency factor expression, tumour cell proliferation and cisplatin-sensitivity in testicular germ cell tumours.

BACKGROUND: Testicular germ cell tumours (TGCTs) are characterised by an overall high cisplatin-sensitivity which has been linked to their continued expression of pluripotency factors. Recently, the Nodal signalling pathway has been implicated in the regulation of pluripotency factor expression in fetal germ cells, and the pathway could therefore also be involved in regulating expression of pluripotency factors in malignant germ cells, and hence cisplatin-sensitivity in TGCTs. METHODS: We used in vitro culture of the TGCT-derived cell line NTera2, ex vivo tissue culture of primary TGCT specimens and xenografting of NTera2 cells into nude mice in order to investigate the consequences of manipulating Nodal and Activin signalling on pluripotency factor expression, apoptosis, proliferation and cisplatin-sensitivity. RESULTS: The Nodal signalling factors were markedly expressed concomitantly with the pluripotency factor OCT4 in GCNIS cells, seminomas and embryonal carcinomas. Despite this, inhibition of Nodal and Activin signalling either alone or simultaneously did not affect proliferation or apoptosis in malignant germ cells in vitro or ex vivo. Interestingly, inhibition of Nodal signalling in vitro reduced the expression of pluripotency factors and Nodal pathway genes, while stimulation of the pathway increased their expression. However, cisplatin-sensitivity was not affected following pharmacological inhibition of Nodal/Activin signalling or siRNA-mediated knockdown of the obligate co-receptor CRIPTO in NTera2 cells in vitro or in a xenograft model. CONCLUSION: Our findings suggest that the Nodal signalling pathway may be involved in regulating pluripotency factor expression in malignant germ cells, but manipulation of the pathway does not appear to affect cisplatin-sensitivity or tumour cell proliferation.

Soluble urokinase plasminogen activator receptor (suPAR) is a novel, independent predictive marker of myocardial infarction in HIV-1-infected patients: a nested case-control study.

OBJECTIVES: Patients infected with HIV are at increased risk of myocardial infarction (MI). Increased plasma levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor (suPAR) have been associated with increased risk of cardiovascular diseases (CVD), including MI in the general population. We tested suPAR as a predictive biomarker of MI in HIV-1-infected individuals. METHODS: suPAR levels were investigated in a nested case-control study of 55 HIV-1-infected cases with verified first-time MI and 182 HIV-1-infected controls with no known CVD. Controls were matched for age, gender, duration of antiretroviral therapy (ART), smoking and no known CVD. suPAR was measured in the four plasma samples available for each patient at different time-points; 1, Before initiation of ART; 2, 3 months after initiation of ART; 3, 1 year before the case's MI; and 4, The last sample available before the case's MI. RESULTS: In unadjusted conditional regression analysis, higher levels of suPAR were associated with a significant increase in risk of MI at all time-points. Patients in the third and fourth suPAR quartiles had a three- to 10-fold higher risk of MI compared to patients in the lowest suPAR quartile at all time-points. suPAR remained a strong significant predictor of MI, when adjusting for HIV-1 RNA, total cholesterol, triglycerides and high-density lipoprotein. CONCLUSION: Elevated suPAR levels were associated with increased risk of MI in HIV-infected patients, suggesting that suPAR could be a useful biomarker for prediction of first-time MI in this patient group, even years before the event.

Associations of subjective vitality with DNA damage, cardiovascular risk factors and physical performance.

AIM: To examine associations of DNA damage, cardiovascular risk factors and physical performance with vitality, in middle-aged men. We also sought to elucidate underlying factors of physical performance by comparing physical performance parameters to DNA damage parameters and cardiovascular risk factors. METHODS: We studied 2487 participants from the Metropolit cohort of 11 532 men born in 1953 in the Copenhagen Metropolitan area. The vitality level was estimated using the SF-36 vitality scale. Cardiovascular risk factors were determined by body mass index (BMI), and haematological biochemistry tests obtained from non-fasting participants. DNA damage parameters were measured in peripheral blood mononuclear cells (PBMCs) from as many participants as possible from a representative subset of 207 participants. RESULTS: Vitality was inversely associated with spontaneous DNA breaks (measured by comet assay) (P = 0.046) and BMI (P = 0.002), and positively associated with all of the physical performance parameters (all P < 0.001). Also, we found several associations between physical performance parameters and cardiovascular risk factors. In addition, the load of short telomeres was inversely associated with maximum jump force (P = 0.018), with lowered significance after exclusion of either arthritis sufferers (P = 0.035) or smokers (P = 0.031). CONCLUSION: Here, we show that self-reported vitality is associated with DNA breaks, BMI and objective (measured) physical performance in a cohort of middle-aged men. Several other associations in this study verify clinical observations in medical practice. In addition, the load of short telomeres may be linked to peak performance in certain musculoskeletal activities.

Thymidine kinase 1 deficient cells show increased survival rate after UV-induced DNA damage.

Balanced deoxynucleotide pools are known to be important for correct DNA repair, and deficiency for some of the central enzymes in deoxynucleotide metabolism can cause imbalanced pools, which in turn can lead to mutagenesis and cell death. Here we show that cells deficient for the thymidine salvage enzyme thymidine kinase 1 (TK1) are more resistant to UV-induced DNA damage than TK1 positive cells although they have thymidine triphosphate (dTTP) levels of only half the size of control cells. Our results suggest that higher thymidine levels in the TK- cells caused by defect thymidine salvage to dTTP protects against UV irradiation.

The role of mitochondrial dNTP levels in cells with reduced TK2 activity.

Both the nuclear and mitochondrial DNA (mtDNA) depend on separate balanced pools of dNTPs for correct function of DNA replication and repair of DNA damage. Import of dNTPs from the cytosolic compartment to the mitochondria has been suggested to have the potential of rectifying a mitochondrial dNTP imbalance. Reduced TK2 activity has been demonstrated to result in mitochondrial dNTP imbalance and consequently mutations of mtDNA in non-dividing cells. In this study, the consequences of a reduced thymidine kinase 2 (TK2) activity were measured in proliferating HeLa cells, on both whole-cell as well as mitochondrial dNTP levels. With the exception of increased mitochondrial dCTP level no significant difference was found in cells with reduced TK2 activity. Our results suggest that import of cytosolic dNTPs in mitochondria of proliferating cells can compensate a TK2 induced imbalance of the mitochondrial dNTP pool.

The role of mismatch repair in small-cell lung cancer cells.

The role of mismatch repair (MMR) in small-cell lung cancer (SCLC) is controversial, as the phenotype of a MMR-deficiency, microsatellite instability (MSI), has been reported to range from 0 to 76%. We studied the MMR pathway in a panel of 21 SCLC cell lines and observed a highly heterogeneous pattern of MMR gene expression. A significant correlation between the mRNA and protein levels was found. We demonstrate that low hMLH1 gene expression was not linked to promoter CpG methylation. One cell line (86MI) was found to be deficient in MMR and exhibited resistance to the alkylating agent MNNG. Surprisingly, MSI was not detected in 86MI and it appears to express all the major MMR components hMSH2, hMSH6, hMLH1, hPMS2, hMSH3, hMLH3, MBD4 (MED1) and hExo1. These data are consistent with at least two possibilities: (1) A missense mutation in one of the MMR genes, which dissociates MSI from drug resistance, or (2) inactivation of a second pathway that leads to MMR-deficiency and MNNG resistance, but induces negligible levels of MSI. We conclude that MMR deficiency is largely not associated with the pathogenesis of SCLC.

Myotonic dystrophy type 2: molecular, diagnostic and clinical spectrum.

BACKGROUND: Myotonic dystrophy types 1 (DM1) and 2 (DM2/proximal myotonic myopathy PROMM) are dominantly inherited disorders with unusual multisystemic clinical features. The authors have characterized the clinical and molecular features of DM2/PROMM, which is caused by a CCTG repeat expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. METHODS: Three-hundred and seventy-nine individuals from 133 DM2/PROMM families were evaluated genetically, and in 234 individuals clinical and molecular features were compared. RESULTS: Among affected individuals 90% had electrical myotonia, 82% weakness, 61% cataracts, 23% diabetes, and 19% cardiac involvement. Because of the repeat tract's unprecedented size (mean approximately 5,000 CCTGs) and somatic instability, expansions were detectable by Southern analysis in only 80% of known carriers. The authors developed a repeat assay that increased the molecular detection rate to 99%. Only 30% of the positive samples had single sizeable expansions by Southern analysis, and 70% showed multiple bands or smears. Among the 101 individuals with single expansions, repeat size did not correlate with age at disease onset. Affected offspring had markedly shorter expansions than their affected parents, with a mean size difference of -17 kb (-4,250 CCTGs). CONCLUSIONS: DM2 is present in a large number of families of northern European ancestry. Clinically, DM2 resembles adult-onset DM1, with myotonia, muscular dystrophy, cataracts, diabetes, testicular failure, hypogammaglobulinemia, and cardiac conduction defects. An important distinction is the lack of a congenital form of DM2. The clinical and molecular parallels between DM1 and DM2 indicate that the multisystemic features common to both diseases are caused by CUG or CCUG expansions expressed at the RNA level.

HNPCC mutations in the human DNA mismatch repair gene hMLH1 influence assembly of hMutLalpha and hMLH1-hEXO1 complexes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common inherited form of neoplasia caused by germline mutations in DNA mismatch repair (MMR) genes. MMR proteins have been reported to associate with several proteins, including the human exonuclease 1 (hEXO1). We report here novel HNPCC-hMLH1 mutant proteins (T117M, Q426X and 1813insA) in Danish HNPCC patients. We demonstrate that these mutant HNPCC-hMLH1 proteins are unable to form complexes with hEXO1 and hPMS2 in vivo. The results indicate that mutations found in HNPCC gene carriers disrupt hMLH1-hEXO1 complex formation and hMutLalpha heterodimer assembly essential for MMR activity.

The LipB protein is a negative regulator of dam gene expression in Escherichia coli.

Transcription initiation of the major promoter (P2) of the Escherichia coli dam gene increases with growth rate. The presence of three partially palindromic motifs, (TTCAGT(N(20))TGAG), designated G (growth)-boxes, within the -52 to +31 region of the promoter, may be related to growth rate control. Deletion of two of these repeats, downstream of the transcription initiation point, result in constitutive high activity of the promoter. The unlinked cde-4::miniTn10 insertion also results in severalfold higher activity of the dam P2 promoter, suggesting that this mutation resulted in the loss of a putative dam P2 repressor. The cde-4 mutation was mapped to the lipB (lipoic acid) gene, which we show encodes a 24 kDa protein initiating at a TTG codon. LipB is a highly conserved protein in animal and plant species, other bacteria, Archaea, and yeast. Plasmids expressing the native or hexahistidine-tagged LipB complement the phenotype of the cde-4 mutant strain. The level of LipB in vivo was higher in exponentially growing cells than those in the stationary phase. Three G-box motifs were also found in the lipB region. Models for the regulation of expression of the two genes are discussed.

Identification of factors interacting with hMSH2 in the fetal liver utilizing the yeast two-hybrid system. In vivo interaction through the C-terminal domains of hEXO1 and hMSH2 and comparative expression analysis.

Mutations in DNA mismatch repair (MMR) genes have been shown to segregate with Hereditary Nonpolyposis Colorectal Cancer (HNPCC). However, because many HNPCC families fail to display mutations in known MMR genes, we argued that changes in other components of the MMR pathway may be responsible. The increasing number of proteins reported to interact in the MMR pathway suggests that larger complexes are formed, the composition of which may differ among cell types and tissues. In an attempt to identify tissue-specific MMR-associated factors, we employed the yeast two-hybrid system, using the human hMSH2 as bait and a human fetal liver library as prey. We demonstrate that hMSH2 interacts with a human 5'-3' exonuclease 1 (hEXO1/HEX1) and that this interaction is mediated through their C-terminal domains. The hMSH6 protein does not interact with hEXO1 in the two-hybrid system. Dot-blot analysis of multiple tissue RNA revealed that hMSH2 and hEXO1 are coexpressed at high levels in fetal liver as well as in adult testis and thymus. Northern blot analysis also revealed that hEXO1/HEX1 is highly expressed in several liver cancer cell lines as well as in colon and pancreas adenocarcinomas, but not in the corresponding non-neoplastic tissue.

The human cyclin B1 protein modulates sensitivity of DNA mismatch repair deficient prostate cancer cell lines to alkylating agents.

DNA damage caused by alkylating agents results in a G2 checkpoint arrest. DNA mismatch repair (MMR) deficient cells are resistant to killing by alkylating agents and are unable to arrest the cell cycle in G2 phase after alkylation damage. We investigated the response of two MMR-deficient prostate cancer cell lines DU145 and LNCaP to the alkylating agent MNNG. Our studies reveal that DU145 cancer cells are more sensitive to killing by MNNG than LNCaP. Investigation of the underlying reasons for lower resistance revealed that the DU145 cells contain low endogenous levels of cyclin B1. We provide direct evidence that the endogenous level of cyclin B1 modulates the sensitivity of MMR-deficient prostate cancer cells to alkylating agents.

Modulation of the gene network connected to interferon-gamma in liver regeneration from oval cells.

Suppression subtractive hybridization was used to clone genes associated with proliferation of oval cells in rat liver regenerating after a 70% partial hepatectomy combined with the feeding of 2-acetylaminofluorene. A subset of the identified genes comprised interferon-gamma receptor alpha subunit (IFN-gammaRalpha), gp91phox, interleukin-1beta (IL-1beta), lymphocyte function-associated molecule-1alpha (LFA-1), eukaryotic initiation factor-2-associated 67-kd protein (eIF-2-associated 67-kd protein), and alpha-fetoprotein, which constitute part of the cellular program modulated by IFN-gamma. Therefore, expression analysis performed by Northern blotting and immunohistochemistry were extended to include IFN-gamma, the IFN-gamma receptor beta subunit (IFN-gammaRbeta), three secondary response genes induced by interaction of IFN-gamma with IFN-gamma receptor complexes, ie, IL-1beta-converting enzyme (ICE), intercellular adhesion molecule-1 (ICAM-1), and urokinase-type plasminogen activator receptor (uPAR), and a cytokine inducing IFN-gamma expression, ie, interleukin-18 (IL-18). The Northern blot analysis showed that all examined genes were modulated when progenitor-like oval cells were activated and recruited for liver regeneration. Immunohistochemistry localized the subunits of the IFN-gamma receptor complex, IFN-gammaRalpha and IFN-gammaRbeta, the secondary response genes uPAR and ICAM-1, the IFN-gamma-inducing factor IL-18, and ICE to the ductular structures of oval cells. In contrast, during liver regeneration after a 70% partial hepatectomy, only modulation of IL-1beta and ICE was observed. Our results, therefore, indicate that IFN-gamma-mediated events may be particularly important when cells in the bile ductules must respond to liver damage by production of ductular oval cells.

Generation of a strong mutator phenotype in yeast by imbalanced base excision repair.

Increased spontaneous mutation is associated with increased cancer risk. Here, by using a model system, we show that spontaneous mutation can be increased several hundred-fold by a simple imbalance between the first two enzymes involved in DNA base excision repair. The Saccharomyces cerevisiae MAG1 3-methyladenine (3MeA) DNA glycosylase, when expressed at high levels relative to the apurinic/apyrimidinic endonuclease, increases spontaneous mutation by up to approximately 600-fold in S. cerevisiae and approximately 200-fold in Escherichia coli. Genetic evidence suggests that, in yeast, the increased spontaneous mutation requires the generation of abasic sites and the processing of these sites by the REV1/REV3/REV7 lesion bypass pathway. Comparison of the mutator activity produced by Mag1, which has a broad substrate range, with that produced by the E. coli Tag 3MeA DNA glycosylase, which has a narrow substrate range, indicates that the removal of endogenously produced 3MeA is unlikely to be responsible for the mutator effect of Mag1. Finally, the human AAG 3-MeA DNA glycosylase also can produce a small (approximately 2-fold) but statistically significant increase in spontaneous mutation, a result which could have important implications for carcinogenesis.

Mammalian DNA repair methyltransferases shield O4MeT from nucleotide excision repair.

O6-Methylguanine (O6MeG) and O4-methylthymine (O4MeT) are potentially mutagenic DNA lesions that cause G:C-->A:T and A:T-->G:C transition mutations by mispairing during DNA replication, and the repair of O6MeG and O4MeT by DNA repair methyltransferases (MTases) is therefore expected to prevent methylation-induced transitions. The efficiency of O6MeG and O4MeT repair by different MTases can vary by several hundred-fold and the aim of this study was to establish the biological consequences of such differences in the efficiency of repair. The ability of three microbial and two mammalian MTases to prevent methylation-induced G:C-->A:T and A:T-->G:C transitions is taken as a measure of their ability to repair O6MeG and O4MeT in vivo respectively. All five MTases give complete protection against G:C-->A:T transitions. However, while the microbial MTases give complete protection against A:T-->G:C transitions, the mammalian MTases actually sensitize cells to A:T-->G:C transitions. We hypothesize that the mammalian MTases bind O4MeT lesions in vivo but that, because they are extremely slow at subsequent methyl transfer, binding shields O4MeT from repair by the nucleotide excision repair pathway. Results are presented to support this hypothesis.

The Escherichia coli MutS DNA mismatch binding protein specifically binds O(6)-methylguanine DNA lesions.

DNA mismatch repair defects in certain cell types confer resistance to the cytotoxic effects of alkylating agents, suggesting that a normally functioning DNA mismatch repair pathway can actually mediate alkylation-induced cell death. In eukaryotic cells this phenomenon is only observed in cells lacking adequate DNA methyltransferase for the repair of O6-methylguanine (O6MeG) DNA lesions. It has been proposed that O6MeG may act as a substrate for DNA mismatch repair when paired with cytosine and when mispaired with thymine and that repeated futile DNA mismatch repair at O6MeG DNA lesions is cytotoxic. Here we show that the Escherichia coli MutS DNA mismatch repair binding protein does indeed bind specifically to O6MeG DNA lesions. In contrast, MutS does not bind DNA containing another O-alkylated base, namely O4-methylthymine, or another kind of modified guanine, namely 8-oxoguanine. These results provide direct biochemical evidence for the involvement of DNA mismatch repair in specifically processing O6MeG DNA lesions.

Growth-rate-dependent transcription initiation from the dam P2 promoter.

Transcription of the dam gene in Escherichia coli is dependent on growth rate. Using single-copy promoter::lacZYA fusions we found that of the five promoter regions which affect dam expression, only the P2 promoter shows growth-rate dependence. The determinants for growth-rate control must lie in the region -52 to +27 relative to the transcription start point.

Novel growth rate control of dam gene expression in Escherichia coli.

Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters. Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence. Promoter P2 is a typical sigma 70 promoter with 18 bp spacing between the -10 and -35 hexamers. Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response. Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein. Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein. We devised a procedure for selection of mutant cells in which dam gene expression was unregulated. One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of beta-galactosidase activity at all growth rates tested. In contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-4 confirming the different modes of regulation. The cde-4::miniTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene.

New cloning vectors for integration in the lambda attachment site attB of the Escherichia coli chromosome.

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage lambda attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the lambda attachment site attP and (ii) a helper plasmid, bearing the lambda int gene, transcribed from the lambda PR promoter under the control of the temperature-sensitive repressor cI857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 degrees C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 degrees C. These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined.

Carbon metabolism regulates expression of the pfl (pyruvate formate-lyase) gene in Escherichia coli.

The anaerobic expression of pfl is reduced both in a strain mutated in the pgi gene and in a pfkA pfkB double mutant strain when cells are grown in medium supplemented with glucose. When cells are grown in medium supplemented with either fructose or pyruvate, no reduction is observed in these strains. The amount of pyruvate in the cells may be responsible for the reduced expression of pfl in the strains mutated in the genes encoding the glycolytic enzymes. Because of the lowered oxygen concentration in the medium, the expression of pfl is induced when an exponentially growing culture enters the stationary phase. This induction is increased when the Casamino Acid concentration is raised 10-fold or when the medium is supplemented with NaCl. Superhelicity of DNA is decreased in a pgi mutant strain grown in medium supplemented with glucose. The superhelicity is also changed, but the opposite way, in a wild-type strain grown in medium supplemented with Casamino Acids at a high concentration or 0.3 M sodium chloride. Our data show that changes in superhelicity do not affect the aerobic expression of pfl but might be important for the anaerobic induction of pfl.