Rad5 and Its Human Homologs, HLTF and SHPRH, Are Novel Interactors of Mismatch Repair.

DNA mismatch repair (MMR) repairs replication errors, and MMR defects play a role in both inherited cancer predisposition syndromes and in sporadic cancers. MMR also recognizes mispairs caused by environmental and chemotherapeutic agents; however, in these cases mispair recognition leads to apoptosis and not repair. Although mutation avoidance by MMR is fairly well understood, MMR-associated proteins are still being identified. We performed a bioinformatic analysis that implicated Saccharomyces cerevisiae Rad5 as a candidate for interacting with the MMR proteins Msh2 and Mlh1. Rad5 is a DNA helicase and E3 ubiquitin ligase involved in post-replicative repair and damage tolerance. We confirmed both interactions and found that the Mlh1 interaction is mediated by a conserved Mlh1-interacting motif (MIP box). Despite this, we did not find a clear role for Rad5 in the canonical MMR mutation avoidance pathway. The interaction of Rad5 with Msh2 and Mlh1 is conserved in humans, although each of the Rad5 human homologs, HLTF and SHPRH, shared only one of the interactions: HLTF interacts with MSH2, and SHPRH interacts with MLH1. Moreover, depletion of SHPRH, but not HLTF, results in a mild increase in resistance to alkylating agents although not as strong as loss of MMR, suggesting gene duplication led to specialization of the MMR-protein associated roles of the human Rad5 homologs. These results provide insights into how MMR accessory factors involved in the MMR-dependent apoptotic response interact with the core MMR machinery and have important health implications into how human cells respond to environmental toxins, tumor development, and treatment choices of tumors with defects in Rad5 homologs.

Erratum: Author Correction: Identification of novel genetic variants predisposing to familial oral squamous cell carcinomas.

[This corrects the article DOI: 10.1038/s41421-019-0126-6.].

Identification of novel genetic variants predisposing to familial oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) is a common subtype of head and neck squamous cell carcinoma (HNSCC), but the pathogenesis underlying familial OSCCs is unknown. Here, we analyzed whole-genome sequences of a family with autosomal dominant expression of oral tongue cancer and identified proto-oncogenes VAV2 and IQGAP1 as the primary factors responsible for oral cancer in the family. These two genes are also frequently mutated in sporadic OSCCs and HNSCCs. Functional analysis revealed that the detrimental variants target tumorigenesis-associated pathways, thus confirming that these novel genetic variants help to establish a predisposition to familial OSCC.

Coronary Artery Disease Risk-Associated Plpp3 Gene and Its Product Lipid Phosphate Phosphatase 3 Regulate Experimental Atherosclerosis.

OBJECTIVE: Genome-wide association studies identified novel loci in PLPP3(phospholipid phosphatase 3) that associate with coronary artery disease risk independently of traditional risk factors. PLPP3 encodes LPP3 (lipid phosphate phosphatase 3), a cell-surface enzyme that can regulate the availability of bioactive lysophopsholipids including lysophosphatidic acid (LPA). The protective allele of PLPP3 increases LPP3 expression during cell exposure to oxidized lipids, however, the role of LPP3 in atherosclerosis remains unclear. Approach and Results: In this study, we sought to validate LPP3 as a determinate of the development of atherosclerosis. In experimental models of atherosclerosis, LPP3 is upregulated and co-localizes with endothelial, smooth muscle cell, and CD68-positive cell markers. Global post-natal reductions in Plpp3 expression in mice substantially increase atherosclerosis, plaque-associated LPA, and inflammation. Although LPP3 expression increases during ox-LDL (oxidized low-density lipoprotein)-induced phenotypic modulation of bone marrow-derived macrophages, myeloid Plpp3 does not appear to regulate lesion formation. Rather, smooth muscle cell LPP3 expression is a critical regulator of atherosclerosis and LPA content in lesions. Moreover, mice with inherited deficiency in LPA receptor signaling are protected from experimental atherosclerosis. CONCLUSIONS: Our results identify a novel lipid signaling pathway that regulates inflammation in the context of atherosclerosis and is not related to traditional risk factors. Pharmacological targeting of bioactive LPP3 substrates, including LPA, may offer an orthogonal approach to lipid-lowering drugs for mitigation of coronary artery disease risk.

Effects of diet and hyperlipidemia on levels and distribution of circulating lysophosphatidic acid.

Lysophosphatidic acids (LPAs) are bioactive radyl hydrocarbon-substituted derivatives of glycerol 3-phosphate. LPA metabolism and signaling are implicated in heritable risk of coronary artery disease. Genetic and pharmacological inhibition of these processes attenuate experimental atherosclerosis. LPA accumulates in atheromas, which may be a consequence of association with LDLs. The source, regulation, and biological activity of LDL-associated LPA are unknown. We examined the effects of experimental hyperlipidemia on the levels and distribution of circulating LPA in mice. The majority of plasma LPA was associated with albumin in plasma from wild-type mice fed normal chow. LDL-associated LPA was increased in plasma from high-fat Western diet-fed mice that are genetically prone to hyperlipidemia (LDL receptor knockout or activated proprotein convertase subtilisin/kexin type 9-overexpressing C57Bl6). Adipose-specific deficiency of the ENPP2 gene encoding the LPA-generating secreted lysophospholipase D, autotaxin (ATX), attenuated these Western diet-dependent increases in LPA. ATX-dependent increases in LDL-associated LPA were observed in isolated incubated plasma. ATX acted directly on LDL-associated lysophospholipid substrates in vitro. LDL from all human subjects examined contained LPA and was decreased by lipid-lowering drug therapies. Human and mouse plasma therefore contains a diet-sensitive LDL-associated LPA pool that might contribute to the cardiovascular disease-promoting effects of LPA.

Regulation of PLPP3 gene expression by NF-κB family transcription factors.

Lipid phosphate phosphatase 3 (LPP3), encoded by the PLPP3 gene, is an integral membrane enzyme that dephosphorylates phosphate esters of glycero- and sphingophospholipids. Cell surface LPP3 can terminate the signaling actions of bioactive lysophosphatidic acid (LPA) and sphingosine 1 phosphate, which likely explains its role in developmental angiogenesis, vascular injury responses, and cell migration. Heritable variants in the final intron PLPP3 associate with interindividual variability in coronary artery disease risk that may result from disruption of enhancer sequences that normally act in cis to increase expression of the gene. However, the mechanisms regulating PLPP3 expression are not well understood. We show that the human PLPP3 promoter contains three functional NF-κB response elements. All of these are required for maximal induction of PLPP3 promoter activity in reporter assays. The identified sequences recruit RelA and RelB components of the NF-κB transcription complex to chromatin, and these transcription factors bind to the identified target sequences in two different cell types. LPA promotes binding of Rel family transcription factors to the PLPP3 promoter and increases PLPP3 gene expression through mechanisms that are attenuated by an NF-κB inhibitor, LPA receptor antagonists, and inhibitors of phosphoinositide 3 kinase. These findings indicate that up-regulation of PLPP3 during inflammation and atherosclerosis results from canonical activation of the NF-κB signaling cascade to increase PLPP3 expression through nuclear import and binding of RelA and RelB transcription factors to the PLPP3 promoter and suggest a mechanism by which the LPP3 substrate, LPA, can regulate PLPP3 expression.

Cancer-driving H3G34V/R/D mutations block H3K36 methylation and H3K36me3-MutSα interaction.

Somatic mutations on glycine 34 of histone H3 (H3G34) cause pediatric cancers, but the underlying oncogenic mechanism remains unknown. We demonstrate that substituting H3G34 with arginine, valine, or aspartate (H3G34R/V/D), which converts the non-side chain glycine to a large side chain-containing residue, blocks H3 lysine 36 (H3K36) dimethylation and trimethylation by histone methyltransferases, including SETD2, an H3K36-specific trimethyltransferase. Our structural analysis reveals that the H3 "G33-G34" motif is recognized by a narrow substrate channel, and that H3G34/R/V/D mutations impair the catalytic activity of SETD2 due to steric clashes that impede optimal SETD2-H3K36 interaction. H3G34R/V/D mutations also block H3K36me3 from interacting with mismatch repair (MMR) protein MutSα, preventing the recruitment of the MMR machinery to chromatin. Cells harboring H3G34R/V/D mutations display a mutator phenotype similar to that observed in MMR-defective cells. Therefore, H3G34R/V/D mutations promote genome instability and tumorigenesis by inhibiting MMR activity.

The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSα.

DNA mismatch repair (MMR) ensures replication fidelity by correcting mismatches generated during DNA replication. Although human MMR has been reconstituted in vitro, how MMR occurs in vivo is unknown. Here, we show that an epigenetic histone mark, H3K36me3, is required in vivo to recruit the mismatch recognition protein hMutSα (hMSH2-hMSH6) onto chromatin through direct interactions with the hMSH6 PWWP domain. The abundance of H3K36me3 in G1 and early S phases ensures that hMutSα is enriched on chromatin before mispairs are introduced during DNA replication. Cells lacking the H3K36 trimethyltransferase SETD2 display microsatellite instability (MSI) and an elevated spontaneous mutation frequency, characteristic of MMR-deficient cells. This work reveals that a histone mark regulates MMR in human cells and explains the long-standing puzzle of MSI-positive cancer cells that lack detectable mutations in known MMR genes.

Coordinated processing of 3' slipped (CAG)n/(CTG)n hairpins by DNA polymerases ß and δ preferentially induces repeat expansions.

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3'-slipped hairpin using its 3'-5' proofreading activity when the hairpin contains no immediate 3' complementary sequences. However, in the presence of pol ß, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol ß incorporates several nucleotides to the hairpin 3'-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3'-slipped (CAG)n/(CTG)n hairpins by polymerases δ and ß on during DNA synthesis induces CAG/CTG repeat expansions.

Modulation of microRNA processing by mismatch repair protein MutLα.

MicroRNAs (miRNAs) are critical post-transcriptional regulators and are derived from hairpin-shaped primary transcripts via a series of processing steps. However, how the production of individual miRNAs is regulated remains largely unknown. Similarly, loss or overexpression of the key mismatch repair protein MutLα (MLH1-PMS2 heterodimer) leads to genome instability and tumorigenesis, but the mechanisms controlling MutLα expression are unknown. Here we demonstrate in vitro and in vivo that MLH1 and miR-422a participate in a feedback loop that regulates the level of both molecules. Using a defined in-vitro miRNA processing system, we show that MutLα stimulates the conversion of pri-miR-422a to pre-miR-422a, as well as the processing of other miRNAs tested, implicating MutLα as a general stimulating factor for miRNA biogenesis. This newly identified MutLα function requires its ATPase and pri-miRNA binding activities. In contrast, miR-422a downregulates MutLα levels by suppressing MLH1 expression through base pairing with the MLH1 3'-untranslated region. A model depicting this feedback mechanism is discussed.

Specific sequence determinants of miR-15/107 microRNA gene group targets.

MicroRNAs (miRNAs) target mRNAs in human cells via complex mechanisms that are still incompletely understood. Using anti-Argonaute (anti-AGO) antibody co-immunoprecipitation, followed by microarray analyses and downstream bioinformatics, 'RIP-Chip' experiments enable direct analyses of miRNA targets. RIP-Chip studies (and parallel assessments of total input mRNA) were performed in cultured H4 cells after transfection with miRNAs corresponding to the miR-15/107 gene group (miR-103, miR-107, miR-16 and miR-195), and five control miRNAs. Three biological replicates were run for each condition with a total of 54 separate human Affymetrix Human Gene 1.0 ST array replicates. Computational analyses queried for determinants of miRNA:mRNA binding. The analyses support four major findings: (i) RIP-Chip studies correlated with total input mRNA profiling provides more comprehensive information than using either RIP-Chip or total mRNA profiling alone after miRNA transfections; (ii) new data confirm that miR-107 paralogs target coding sequence (CDS) of mRNA; (iii) biochemical and computational studies indicate that the 3' portion of miRNAs plays a role in guiding miR-103/7 to the CDS of targets; and (iv) there are major sequence-specific targeting differences between miRNAs in terms of CDS versus 3'-untranslated region targeting, and stable AGO association versus mRNA knockdown. Future studies should take this important miRNA-to-miRNA variability into account.

The miR-15/107 group of microRNA genes: evolutionary biology, cellular functions, and roles in human diseases.

The miR-15/107 group of microRNA (miRNA) gene is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease and neurodegenerative disease, including Alzheimer's disease. Here we provide an overview of the following: (1) the evolution of miR-15/107 group member genes; (2) the expression levels of miRNAs in mammalian tissues; (3) evidence for overlapping gene-regulatory functions by different miRNAs; (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs; and (5) the roles played by these miRNAs in human diseases. Membership in this group is defined based on sequence similarity near the mature miRNAs' 5' end: all include the sequence AGCAGC. Phylogeny of this group of miRNAs is incomplete; thus, a definitive taxonomic classification (e.g., designation as a "superfamily") is currently not possible. While all vertebrates studied to date express miR-15a, miR-15b, miR-16, miR-103, and miR-107, mammals alone are known to express miR-195, miR-424, miR-497, miR-503, and miR-646. Multiple different miRNAs in the miR-15/107 group are expressed at moderate to high levels in human tissues. We present data on the expression of all known miR-15/107 group members in human cerebral cortical gray matter and white matter using new miRNA profiling microarrays. There is extensive overlap in the mRNAs targeted by miR-15/107 group members. We show new data from cultured H4 cancer cells that demonstrate similarities in mRNAs targeted by miR-16 and miR-103 and also support the importance of the mature miRNAs' 5' seed region in mRNA target recognition. In conclusion, the miR-15/107 group of miRNA genes is a fascinating topic of study for evolutionary biologists, miRNA biochemists, and clinically oriented translational researchers alike.

Altered 8-oxoguanine glycosylase in mild cognitive impairment and late-stage Alzheimer's disease brain.

Eight-hydroxy-2'-deoxyguanosine (8-OHdG) is increased in the brain in late-stage Alzheimer's disease (LAD) and mild cognitive impairment (MCI). To determine if decreased base-excision repair contributes to these elevations, we measured oxoguanine glycosylase 1 (OGG1) protein and incision activities in nuclear and mitochondrial fractions from frontal (FL), temporal (TL), and parietal (PL) lobes from 8 MCI and 7 LAD patients, and 6 age-matched normal control (NC) subjects. OGG1 activity was significantly (P<0.05) decreased in nuclear specimens of FL, TL, and PL in MCI and LAD and in mitochondria from LAD FL and TL and MCI TL. Nuclear OGG1 protein was significantly decreased in LAD FL and MCI and LAD PL. No differences in mitochondrial OGG1 protein levels were found. Overall, our results suggest that decreased OGG1 activity occurs early in the progression of AD, possibly mediated by 4-hydroxynonenal inactivation and may contribute to elevated 8-OHdG in the brain in MCI and LAD.

Preferential loss of mismatch repair function in refractory and relapsed acute myeloid leukemia: potential contribution to AML progression.

Acute myeloid leukemia (AML) is an aggressive hematological cancer. Despite therapeutic regimens that lead to complete remission, the vast majority of patients undergo relapse. The molecular mechanisms underlying AML development and relapse remain incompletely defined. To explore whether loss of DNA mismatch repair (MMR) function is involved in AML, we screened two key MMR genes, MSH2 and MLH1, for mutations and promoter hypermethylation in leukemia specimens from 53 AML patients and blood from 17 non-cancer controls. We show here that whereas no amino acid alteration or promoter hypermethylation was detected in all control samples, 18 AML patients exhibited either mutations in MMR genes or hypermethylation in the MLH1 promoter. In vitro functional MMR analysis revealed that almost all the mutations analyzed resulted in loss of MMR function. MMR defects were significantly more frequent in patients with refractory or relapsed AML compared with newly diagnosed patients. These observations suggest for the first time that the loss of MMR function is associated with refractory and relapsed AML and may contribute to disease pathogenesis.

Evidence that a mutation in the MLH1 3'-untranslated region confers a mutator phenotype and mismatch repair deficiency in patients with relapsed leukemia.

Defects in DNA mismatch repair (MMR) are the molecular basis of certain cancers, including hematological malignancies. The defects are often caused by mutations in coding regions of MMR genes or promoter methylation of the genes. However, in many cases, despite that a hypermutable phenotype is detected in a patient, no mutations/hypermethylations of MMR genes can be detected. We report here a novel mechanism that a mutation in the MLH1 3'-untranslated region (3'-UTR) leads to MMR deficiency. A relapsed leukemia patient displayed microsatellite instability, but no genetic and epigenetic alterations in key MMR genes were identifiable. Instead, a 3-nucleotide (TTC) deletion in the MLH1 3'-UTR was found in the patient's blood sample. The mutant MLH1 3'-UTR was found to significantly reduce the expressions of both a firefly luciferase reporter gene and an ectopic MLH1 gene in model cell lines. Consistent with these observations, a significant reduction in the steady-state level of MLH1 mRNA was observed in white blood cells of the patient. These findings suggest that the mutant MLH1 3'-UTR can cause a severely reduced/defective MMR activity conferring leukemia relapse, likely by down-regulating MLH1 expression at the mRNA level. Although the exact mechanism by which the mutant 3'-UTR down-regulates the MLH1 mRNA is not known, our findings provide a novel marker for cancers with MMR defects.

Identification and characterization of OGG1 mutations in patients with Alzheimer's disease.

Patients with Alzheimer's disease (AD) exhibit higher levels of 8-oxo-guanine (8-oxoG) DNA lesions in their brain, suggesting a reduced or defective 8-oxoG repair. To test this hypothesis, this study investigated 14 AD patients and 10 age-matched controls for mutations of the major 8-oxoG removal gene OGG1. Whereas no alterations were detected in any control samples, four AD patients exhibited mutations in OGG1, two carried a common single base (C796) deletion that alters the carboxyl terminal sequence of OGG1, and the other two had nucleotide alterations leading to single amino acid substitutions. In vitro biochemical assays revealed that the protein encoded by the C796-deleted OGG1 completely lost its 8-oxoG glycosylase activity, and that the two single residue-substituted OGG1 proteins showed a significant reduction in the glycosylase activity. These results were consistent with the fact that nuclear extracts derived from a limited number of AD patients with OGG1 mutations exhibited greatly reduced 8-oxoG glycosylase activity compared with age-matched controls and AD patients without OGG1 alterations. Our findings suggest that defects in OGG1 may be important in the pathogenesis of AD in a significant fraction of AD patients and provide new insight into the molecular basis for the disease.

Effect of alpha-pinene on nuclear translocation of NF-kappa B in THP-1 cells.

AIM: To study the effects of alpha-pinene on nuclear translocation of nuclear factor-kappa B (NF-kappa B) and the expression of the inhibitor of NF-kappa B (I kappa B alpha) in human monocyte THP-1 cell line. METHODS: THP-1 cells were incubated with alpha-pinene (1, 10, and 100 mg/L, for 30 min) before being stimulated with lipopolysaccharide (LPS, 1 mg/L, 30 min). The location of NF-kappa B p65 subunit (NF-kappa B/p65) in THP-1 cells was detected by immunofluorescence and laser scanning confocal microscope (LSCM). The expression of NF-kappa B/p65 in nuclei and that of I kappa B alpha in cytoplasm were measured by Western-blot analysis. RESULTS: The majority of FITC-labelled NF-kappa B/p65 was located in the nuclei being stimulated with LPS. Whereas, no such fluorescence was seen in the nuclei of the groups pretreated with alpha-pinene or control cells. alpha-Pinene pretreatment decreased the NF-kappa B/p65 nuclear translocation in LPS-stimulated THP-1 cells, and this effect was dose-dependent, but there was no reaction in LPS-unstimulated THP-1 cells. alpha-Pinene pretreatment increased I kappa B alpha protein level in cytoplasm, compared with that in LPS-stimulated THP-1 cells. CONCLUSION: In a dose-related fashion, alpha-pinene inhibits the nuclear translocation of NF-kappa B induced by LPS in THP-1 cells, and this effect is partly due to the upregulation of I kappa B alpha expression.

[Effect of eucalyptus globulus oil on activation of nuclear factor-kappaB in THP-1 cells].

OBJECTIVE: To study the effect of eucalyptus globulus oil on the activity of nuclear factor-kappaB(NF-kappaB) in THP-1 cell line. METHODS: THP-1 cells were cultured with or without eucalyptus globulus oil at different concentrations (1, 10, 100 mg x L(-1), 30 min) before being stimulated with lipopolysaccharide (LPS, 1 mg x L(-1), 30 min). The location of NF-kappaB p65 subunit (NF-kappaB/p65) in THP-1 cells was detected by indirect immunofluorescence and laser scanning confocal microscope. The expression of NF-kappaB/p65 in nuclei was measured by Western-blot analysis. RESULT: The FITC-label NF-kappaB/p65 was mainly located in the nuclei after THP-1 cells were stimulated with LPS. Whereas, no fluorescence were seen in the nuclei of cells pretreated with eucalyptus globulus oil. This effect on NF-kappaB/p65 nuclear translocation was in a concentration dependent manner. CONCLUSION: Eucalyptus globulus oil inhibits the nuclear translocation of NF-kappaB induced by LPS in THP-1 cells.

Purification and relationship with gastric disease of a 130 kDa (CagA) protein of Helicobacter pylori.

OBJECTIVE: The aims of this research were to purify and identify the 130 kDa (CagA) protein of H. pylori clinical isolate HP97002 and evaluate the relationships between the purified 130 kDa (CagA) protein and gastric diseases. METHODS: The procedure for isolating the protein included 6 mol/L guanidine extract, size exclusion chromatography and elusion from gel. Sera of 68 patients with gastric diseases (44 with chronic gastritis, 15 with atrophic gastritis, 7 with peptic ulcer disease, 2 with gastric cancer) were obtained, and the serological response to CagA was studied by Western-blot using the purified protein. RESULTS: The purified protein was 130 kDa and preserved good antigenicity and revealed basic isoelectric point about of 8.1. Among 68 sera, 43 sera could recognize the purified protein associated with chronic gastritis 47.7% (21/44), atrophic gastritis 86.7% (13/15), peptic ulcer disease 100% (77), gastric cancer 100% (2/2). Compared with each other, the difference was significant (chi2 = 13.327, P = 0.004), and 130 kDa (CagA) protein was associated with severe gastric diseases ( r(S) = 0.442, P = 0.001). CONCLUSION: The 130 kDa (CagA) protein was associated with severe gastric diseases.

[The effect of silicon dioxide on the activation of nuclear factor-kappaB in THP-1 cells].

OBJECTIVE: To study the effect of silicon dioxide (SiO(2)) on the activation of nuclear factor-kappaB (NF-kappaB) in THP-1 cell line. METHODS: THP-1 cells were incubated with a series of doses of SiO(2) (0, 100, 200 micro g/ml). The location of NF-kappaB p65 subunit (NF-kappaB/p65) in THP-1 cells was detected by immunofluorescence and laser scanning confocal microscope (LSCM). The expression of NF-kappaB/p65 in nuclei was measured by Western blot analysis. RESULTS: The majority of fluorescein isothiocyanate (FITC)-labelled NF-kappaB/p65 located in the nuclei 30 min after stimulation by 100 micro g/ml SiO(2), whereas the FITC-labelled NF-kappaB/p65 were mainly seen in the plasma of normal control cells. The expression of NF-kappaB/p65 in THP-1 nuclear protein was low in control group (0 micro g/ml SiO(2)) while it increased after stimulation by 100 micro g/ml SiO(2) and 200 micro g/ml SiO(2) for 15 min and 30 min. The level of NF-kappaB/p65 was comparatively increased with the increasing of doses and time. Lipopolysaccharides (LPS), an activator of NF-kappaB, had similar effect as SiO(2) on the activation of NF-kappaB/p65 in THP-1 cells. CONCLUSION: SiO(2) could activate and internalize NF-kappaB in the THP-1 cell line.